Polypeptides, antibody variable domains and antagonists

ABSTRACT

The invention relates to anti-VEGF polypeptides and antibody single variable domains (dAbs) that are resistant to degradation by a protease, as well as antagonists comprising these. The polypeptides, dAbs and antagonists are useful for pulmonary administration, oral administration, delivery to the lung and delivery to the GI tract of a patient, as well as for treating cancer and inflammatory disease, such as arthritis.

RELATED APPLICATIONS

This application is the US National Stage application, made under 35 USC§371, of International Application No. PCT/GB2008/050404, filed Jun. 3,2008 and published in English which claims priority under 35 USC §119,or 35 USC §365, to U.S. Provisional Application No. 60/933,632 filedJun. 6, 2007 and United Kingdom, Application No. 0724331.4, filed Dec.13, 2007. The entire teachings of the above applications areincorporated herein by reference.

The present invention relates to protease resistant polypeptides,immunoglobulin (antibody) single variable domains and vascularendothelial growth factor (VEGF) antagonists comprising these. Theinvention further relates to uses, formulations, compositions anddevices comprising such anti-VEGF ligands.

BACKGROUND OF THE INVENTION

Polypeptides and peptides have become increasingly important agents in avariety of applications, including industrial applications and use asmedical, therapeutic and diagnostic agents. However, in certainphysiological states, such as Cancer and inflammatory states (e.g.,COPD), the amount of proteases present in a tissue, organ or animal(e.g., in the lung, in or adjacent to a tumor) can increase. Thisincrease in proteases can result in accelerated degradation andinactivation of endogenous proteins and of therapeutic peptides,polypeptides and proteins that are administered to treat disease.Accordingly, some agents that have potential for in vivo use (e.g., usein treating, diagnosing or preventing disease) have only limitedefficacy because they are rapidly degraded and inactivated by proteases.

Protease resistant polypeptides provide several advantages. For example,protease resistant polypeptides remaining active in vivo longer thanprotease sensitive agents and, accordingly, remaining functional for aperiod of time that is sufficient to produce biological effects. A needalso exists for improved methods to select polypeptides that areresistant to protease degradation and also have desirable biologicalactivity.

VEGF:

VEGF is a secreted, heparin-binding, homodimeric glycoprotein existingin several alternate forms due to alternative splicing of its primarytranscript (Leung et al., 1989, Science 246: 1306). VEGF is also knownas vascular permeability factor (VPF) due to its ability to inducevascular leakage, a process important in inflammation.

An important pathophysiological process that facilitates tumorformation, metastasis and recurrence is tumor angiogenesis. This processis mediated by the elaboration of angiogenic factors expressed by thetumor, such as VEGF, which induce the formation of blood vessels thatdeliver nutrients to the tumor. Accordingly, an approach to treatingcertain cancers is to inhibit tumor angiogenesis mediated by VEGF,thereby starving the tumor. Avastin (bevacizumab; Genetech, Inc.) is ahumanized antibody that binds human VEGF that has been approved fortreating colorectal cancer. An antibody referred to as antibody 2C3(ATCC Accession No. PTA 1595) is reported to bind VEGF and inhibitbinding of VEGF to epidermal growth factor receptor 2.

Targeting VEGF with currently available therapeutics is not effective inall patients, or for all cancers. Thus, a need exists for improvedagents for treating cancer and other pathological conditions nediated byVEGF e.g. vascular proliferative diseases (e.g. Age related maculardegeneration (AMD)).

VEGF has also been implicated in inflammatory disorders and autoimmunediseases. For example, the identification of VEGF in synovial tissues ofRA patients highlighted the potential role of VEGF in the pathology ofRA (Fava et al., 1994, J. Exp. Med. 180:341:346; Nagashima et al., 1995,J. Rheumatol. 22: 1624-1630). A role for VEGF in the pathology of RA wassolidified following studies in which anti-VEGF antibodies wereadministered in the murine collagen-induced arthritis (CIA) model. Inthese studies, VEGF expression in the joints increased upon induction ofthe disease, and the administration of anti-VEGF antisera blocked thedevelopment of arthritic disease and ameliorated established disease(Sone et al., 2001, Biochem. Biophys. Res. Comm. 281: 562-568; Lu etal., 2000, J. Immunol. 164: 5922-5927). Hence targeting VEGF may also beof benefit in treating RA, and other conditions e.g. those associatedwith inflammation and/or autoimmune disease.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a polypeptide comprising an aminoacid sequence that is at least 97% identical to the amino acid sequenceof DOM15-26-593 (shown in FIG. 5). In one embodiment, the percentidentity is at least 98 or 99%. In one embodiment, the polypeptide isDOM15-26-593. The invention further provides (substantially) pureDOM15-26-593 monomer. In one embodiment, the DOM15-26-593 is at least98, 99, 99.5% pure or 100% pure monomer.

In one aspect, the invention provides a polypeptide (e.g. that isprotease resistant) and that is encoded by an amino acid sequence thatis at least 80% identical to the amino acid sequence of DOM15-26-593(shown in FIG. 5). In one embodiment, the percent identity is at least70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. In one embodimentthat protease resistant polypeptide is obtainable by the methoddescribed herein for isolating protease resistant polypeptides.

In one aspect, the invention provides a polypeptide encoded by an aminoacid sequence that is at least 55% identical to the nucleotide sequenceof the nucleotide sequence of DOM15-26-593 and wherein the polypeptidecomprises an amino acid sequence that is at least 97% identical to theamino acid s the amino acid sequence of DOM15-26-593. In one embodiment,the percent identity of the nucleotide sequence is at least 60, 65, 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. In oneembodiment, the percent identity of the amino acid sequence is at least,98 or 99% or 100%. For example, the nucleotide sequence may be acodon-optimised version of the nucleotide sequence of DOM15-26-593.Codon optimization of sequences is known in the art. In one embodiment,the nucleotide sequence is optimized for expression in a bacterial (eg,E. coli or Pseudomonas, eg P. fluorescens), mammalian (eg, CHO) or yeasthost cell (eg. Pichia or Saccharomyces, eg P. pastoris or S.cerevisiae).

In one aspect, the invention provides a fusion protein comprising thepolypeptide of the invention.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain comprising an amino acid sequence that is at least 97%identical to the amino acid sequence of DOM15-26-593. In one embodiment,the percent identity is at least 98 or 99%.

In one embodiment, the immunoglobulin single variable domain comprisesvaline at position 6, wherein numbering is according to Kabat(“Sequences of Proteins of Immunological Interest”, US Department ofHealth and Human Services 1991).

In one embodiment, the immunoglobulin single variable domain comprisesleucine at position 99, wherein numbering is according to Kabat.

In one embodiment, the immunoglobulin single variable domain comprisesLysine at position 30, wherein numbering is according to Kabat.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain comprising an amino acid sequence that is identical tothe amino acid sequence of DOM15-26-593.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain encoded by a nucleotide sequence that is at least 80%identical to the nucleotide sequence of DOM15-26-593. In one embodiment,the percent identity is at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99%.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain encoded by an amino acid sequence that is at least 55%identical to the nucleotide sequence of the nucleotide sequence ofDOM15-26-593 and wherein the variable domain comprises an amino acidsequence that is at least 97% identical to the amino acid sequence ofDOM15-26-593. In one embodiment, the percent identity of the nucleotidesequence is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99%. In one embodiment, the percent identity of the amino acidsequence is at least 98 or 99% or 100%. For example, the nucleotidesequence may be a codon-optimised version of the nucleotide sequence ofDOM15-26-593. Codon optimization of sequences is known in the art. Inone embodiment, the nucleotide sequence is optimized for expression in abacterial (eg, E. coli or Pseudomonas, eg P. fluorescens), mammalian(eg, CHO) or yeast host cell (eg. Pichia or Saccharomyces, eg P.pastoris or S. cerevisiae).

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain encoded by a sequence that is identical to thenucleotide sequence of DOM15-26-593.

In one aspect, the invention provides an anti-VEGF antagonist comprisingan anti-VEGF immunoglobulin single variable domain according to theinvention. In one embodiment, the antagonist comprises first and secondimmunoglobulin single variable domains, wherein each variable domain isaccording to invention. For example, wherein the antagonist comprises amonomer of said single variable domain or a homodimer of said singlevariable domain. In one embodiment, the amino acid sequence of the oreach single variable domain is identical to the amino acid sequence ofDOM15-26-593.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain comprising an amino acid sequence that is identical tothe amino acid sequence of DOM15-26-593 or differs from the amino acidsequence of DOM15-26-593 at no more than 14 amino acid positions and hasa CDR1 sequence that is at least 50% identical to the CDR1 sequence ofDOM15-26-593. In one embodiment, the difference is no more than 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid position. In oneembodiment, the CDR sequence identity is at least 55, 60, 65, 70, 75,80, 85, 90, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain comprising an amino acid sequence that is identical tothe amino acid sequence of DOM15-26-593 or differs from the amino acidsequence of DOM15-26-593 at no more than 14 amino acid positions and hasa CDR2 sequence that is at least 50% identical to the CDR2 sequence ofDOM15-26-593. In one embodiment, the difference is no more than 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid position.

In one embodiment, the CDR sequence identity is at least 55, 60, 65, 70,75, 80, 85, 90, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain comprising an amino acid sequence that is identical tothe amino acid sequence of DOM15-26-593 or differs from the amino acidsequence of DOM15-26-593 at no more than 14 amino acid positions and hasa CDR3 sequence that is at least 50% identical to the CDR3 sequence ofDOM15-26-593. In one embodiment, the difference is no more than 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid position. In oneembodiment, the CDR sequence identity is at least 55, 60, 65, 70, 75,80, 85, 90, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain comprising an amino acid sequence that is identical tothe amino acid sequence of DOM15-26-593 or differs from the amino acidsequence of DOM15-26-593 at no more than 14 amino acid positions and hasa CDR1 sequence that is at least 50% identical to the CDR1 sequence ofDOM15-26-593 and has a CDR2 sequence that is at least 50% identical tothe CDR2 sequence of DOM15-26-593. In one embodiment, the difference isno more than 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidposition. In one embodiment, one or both CDR sequence identities isrespectively at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or99%.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain comprising an amino acid sequence that is identical tothe amino acid sequence of DOM15-26-593 or differs from the amino acidsequence of D DOM15-26-593 at no more than 14 amino acid positions andhas a CDR1 sequence that is at least 50% identical to the CDR1 sequenceof DOM15-26-593 and has a CDR3 sequence that is at least 50% identicalto the CDR3 sequence of DOM15-26-593. In one embodiment, the differenceis no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidposition. In one embodiment, one or both CDR sequence identities isrespectively at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or99%.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain comprising an amino acid sequence that is identical tothe amino acid sequence of DOM15-26-593 or differs from the amino acidsequence of DOM15-26-593 at no more than 14 amino acid positions and hasa CDR2 sequence that is at least 50% identical to the CDR2 sequence ofDOM15-26-593 and has a CDR3 sequence that is at least 50% identical tothe CDR3 sequence of DOM15-26-593. In one embodiment, the difference isno more than 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidposition. In one embodiment, one or both CDR sequence identities isrespectively at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or99%.

In one aspect, the invention provides an anti-VEGF immunoglobulin singlevariable domain comprising an amino acid sequence that is identical tothe amino acid sequence of DOM15-26-593 or differs from the amino acidsequence of DOM15-26-593 at no more than 14 amino acid positions and hasa CDR1 sequence that is at least 50% identical to the CDR1 sequence ofDOM15-26-593 and has a CDR2 sequence that is at least 50% identical tothe CDR2 sequence of DOM15-26-593 and has a CDR3 sequence that is atleast 50% identical to the CDR3 sequence of DOM15-26-593. In oneembodiment, the difference is no more than 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2 or 1 amino acid position. In one embodiment, one or two oreach CDR sequence identity is at least 55, 60, 65, 70, 75, 80, 85, 90,95, 96, 97, 98 or 99%.

In one aspect, the invention provides a anti-VEGF antagonist having aCDR1 sequence that is at least 50% identical to the CDR1 sequence ofDOM15-26-593. In one embodiment, the CDR sequence identity is at least55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%. The antagonistmay be resistant to protease, for example one or more of the proteasesas herein described, for example under a set of conditions as hereindescribed.

In one aspect, the invention provides an anti-VEGF antagonist having aCDR2 sequence that is at least 50% identical to the CDR1 sequence ofDOM15-26-593. In one embodiment, the CDR sequence identity is at least55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%. The antagonistmay be resistant to protease, for example one or more of the proteasesas herein described, for example under a set of conditions as hereindescribed.

In one aspect, the invention provides an anti-VEGF antagonist having aCDR3 sequence that is at least 50% identical to the CDR1 sequence ofDOM15-26-593. In one embodiment, the CDR sequence identity is at least55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%. The antagonistmay be resistant to protease, for example one or more of the proteasesas herein described, for example under a set of conditions as hereindescribed.

In one aspect, the invention provides an anti-VEGF antagonist having aCDR1 sequence that is at least 50% identical to the CDR1 sequence ofDOM15-26-593 and a CDR2 sequence that is at least 50% identical to theCDR2 sequence of DOM15-26-593. In one embodiment, the CDR sequenceidentity of one or both CDRs is at least 55, 60, 65, 70, 75, 80, 85, 90,95, 96, 97, 98 or 99%. The antagonist may be resistant to protease, forexample one or more of the proteases as herein described, for exampleunder a set of conditions as herein described.

In one aspect, the invention provides an anti-VEGF antagonist having aCDR1 sequence that is at least 50% identical to the CDR1 sequence ofDOM15-26-593 and a CDR3 sequence that is at least 50% identical to theCDR3 sequence of DOM15-26-59. In one embodiment, the CDR sequenceidentity of one or both CDRs is at least 55, 60, 65, 70, 75, 80, 85, 90,95, 96, 97, 98 or 99%. The antagonist may be resistant to protease, forexample one or more of the proteases as herein described, for exampleunder a set of conditions as herein described.

In one aspect, the invention provides an anti-VEGF antagonist having aCDR2 sequence that is at least 50% identical to the CDR2 sequence ofDOM15-26-593 and a CDR3 sequence that is at least 50% identical to theCDR3 sequence of DOM15-26-593. In one embodiment, the CDR sequenceidentity of one or both CDRs is at least 55, 60, 65, 70, 75, 80, 85, 90,95, 96, 97, 98 or 99%. The antagonist may be resistant to protease, forexample one or more of the proteases as herein described, for exampleunder a set of conditions as herein described.

In one aspect, the invention provides an anti-VEGF antagonist having aCDR1 sequence that is at least 50% identical to the CDR1 sequence ofDOM15-26-593 and a CDR2 sequence that is at least 50% identical to theCDR2 sequence of DOM15-26-593 and a CDR3 sequence that is at least 50%identical to the CDR3 sequence of DOM15-26-593. In one embodiment, theCDR sequence identity of one or two or each of the CDRs is at least 55,60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%. The antagonist may beresistant to protease, for example one or more of the proteases asherein described, for example under a set of conditions as hereindescribed.

In one aspect, the invention provides an anti-VEGF antagonist comprisingan immunoglobulin single variable domain comprising the sequence ofCDR1, CDR2, and/or CDR3 (eg, CDR1, CDR2, CDR3, CDR1 and 2, CDR1 and 3,CDR2 and 3 or CDR1, 2 and 3) of DOM15-26-593. The antagonist may beresistant to protease, for example one or more of the proteases asherein described, for example under a set of conditions as hereindescribed.

In one aspect, the invention provides an anti-VEGF antagonist thatcompetes with DOM15-26-593 for binding to VEGF. Thus, the antagonist maybind the same epitope as DOM15-26-593 or an overlapping epitope. In oneembodiment, the antagonist comprises an immunoglobulin single variabledomain having an amino acid sequence that is at least 97% identical tothe amino acid sequence of DOM15-26-593. In one embodiment, the percentidentity is at least 98 or 99%. In one embodiment, the variable domainis DOM15-26-593. The antagonist may be resistant to protease, forexample one or more of the proteases as herein described, for exampleunder a set of conditions as herein described. In one embodiment, theantagonist is an antibody or antigen-binding fragment thereof, such as amonovalent antigen-binding fragment (e.g., scFv, Fab, Fab′, dAb) thathas binding specificity for VEGF. Other examples of antagonists areligands described herein that bind VEGF. The ligands may comprise animmunoglobulin single variable domain or domain antibody (dAb) that hasbinding specificity for VEGF, or the complementarity determining regionsof such a dAb in a suitable format. In some embodiments, the ligand is adAb monomer that consists essentially of, or consists of, animmunoglobulin single variable domain or dAb that has bindingspecificity for VEGF. In other embodiments, the ligand is a polypeptidethat comprises a dAb (or the CDRs of a dAb) in a suitable format, suchas an antibody format.

These VEGF ligands e.g. dAbs, can be formatted to have a largerhydrodynamic size, for example, by attachment of a PEG group, serumalbumin, transferrin, transferrin receptor or at least thetransferrin-binding portion thereof, an antibody Fc region, or byconjugation to an antibody domain. For example, an agent (e.g.,polypeptide, variable domain or antagonist) that i) binds VEGF (ii)antagonizes the activation of VEGF mediated signal transduction, and(iii) does not inhibit the binding of VEGF to its receptor, such as adAb monomer, can be formatted as a larger antigen-binding fragment of anantibody (e.g., formatted as a Fab, Fab′, F(ab)₂, F(ab′)₂, IgG, scFv).The hydrodynaminc size of a ligand and its serum half-life can also beincreased by conjugating or linking a VEGF binding agent (antagonist,variable i) to a binding domain (e.g., antibody or antibody fragment)that binds an antigen or epitope that increases half-live in vivo, asdescribed herein (see, Annex 1 of WO2006038027 incorporated herein byreference in its entirety). For example, the VEGF binding agent (e.g.,polypeptide, E.G. dAb) can be conjugated or linked to an anti-serumalbumin or anti-neonatal Fc receptor antibody or antibody fragment, egan anti-SA or anti-neonatal Fc receptor dAb, Fab, Fab′ or scFv, or to ananti-SA affibody or anti-neonatal Fc receptor affibody.

Examples of suitable albumin, albumin fragments or albumin variants foruse in a VEGF-binding ligands according to the invention are describedin WO 2005/077042A2 and WO2006038027, which are incorporated herein byreference in their entirety.

In other embodiments of the invention described throughout thisdisclosure, instead of the use of a “dAb” in an antagonist or ligand ofthe invention, it is contemplated that the skilled addressee can use adomain that comprises the CDRs of a dAb that binds VEGF (e.g., CDRsgrafted onto a suitable protein scaffold or skeleton, eg an affibody, anSpA scaffold, an LDL receptor class A domain or an EGF domain) or can bea protein domain comprising a binding site for VEGF, e.g., wherein thedomain is selected from an affibody, an SpA domain, an LDL receptorclass A domain or an EGF domain. The disclosure as a whole is to beconstrued accordingly to provide disclosure of antagonists, ligands andmethods using such domains in place of a dAb.

Polypeptides, immunoglobulin single variable domains and antagonists ofthe invention may be resistant to one or more of the following: serineprotease, cysteine protease, aspartate proteases, thiol proteases,matrix metalloprotease, carboxypeptidase (e.g., carboxypeptidase A,carboxypeptidase B), trypsin, chymotrypsin, pepsin, papain, elastase,leukozyme, pancreatin, thrombin, plasmin, cathepsins (e.g., cathepsinG), proteinase (e.g., proteinase 1, proteinase 2, proteinase 3),thermolysin, chymosin, enteropeptidase, caspase (e.g., caspase 1,caspase 2, caspase 4, caspase 5, caspase 9, caspase 12, caspase 13),calpain, ficain, clostripain, actinidain, bromelain, and separase. Inparticular embodiments, the protease is trypsin, elastase or leucozyme.The protease can also be provided by a biological extract, biologicalhomogenate or biological preparation. In one embodiment, the protease isa protease found in sputum, mucus (e.g., gastric mucus, nasal mucus,bronchial mucus), bronchoalveolar lavage, lung homogenate, lung extract,pancreatic extract, gastric fluid, saliva. In one embodiment, theprotease is one found in the eye and/or tears. Examples of suchproteases found in the eye include caspases, calpains, matricmetalloproteases, disintegrin, metalloproteinases (ADAMs) and ADAM withthrombospondin mitifs, the proteosomes, tissue plasminogen activator,secretases, cathepsin B and D, cystatin C, serine protease PRSS1,ubiquitin proteosome pathway (UPP). In one embodiment, the protease is anon-bacterial protease. In an embodiment, the protease is an animal, eg,mammalian, eg, human, protease. In an embodiment, the protease is a GItract protease or a pulmonary tissue protease, eg, a GI tract proteaseor a pulmonary tissue protease found in humans. Such protease listedhere can also be used in the methods described herein involving exposureof a repertoire of library to a protease.

In one aspect, the invention provides a protease resistantimmunoglobulin single variable domain comprising a VEGF binding site,wherein the variable domain is resistant to protease when incubated with

(i) a concentration (c) of at least 10 micrograms/ml protease at 37° C.for time (t) of at least one hour; or

(ii) a concentration (c′) of at least 40 micrograms/ml protease at 30°C. for time (t) of at least one hour. In one embodiment, the ratio (on amole/mole basis) of protease, eg trypsin, to variable domain is 8,000 to80,000 protease:variable domain, eg when C is 10 micrograms/ml, theratio is 800 to 80,000 protease:variable domain; or when C or C′ is 100micrograms/ml, the ratio is 8,000 to 80,000 protease:variable domain. Inone embodiment the ratio (on a weight/weight, eg microgram/microgrambasis) of protease (eg, trypsin) to variable domain is 16,000 to 160,000protease:variable domain eg when C is 10 micrograms/ml, the ratio is1,600 to 160,000 protease:variable domain; or when C or C′ is 100micrograms/ml, the ratio is 1,6000 to 160,000 protease:variable domain.In one embodiment, the concentration (c or c′) is at least 100 or 1000micrograms/ml protease. In one embodiment, the concentration (c or c′)is at least 100 or 1000 micrograms/ml protease. Reference is made to thedescription herein of the conditions suitable for proteolytic activityof the protease for use when working with repertoires or libraries ofpeptides or polypeptides (eg, w/w parameters). These conditions can beused for conditions to determine the protease resistance of a particularimmunoglobulin single variable domain. In one embodiment, time (t) is oris about one, three or 24 hours or overnight (e.g., about 12-16 hours).In one embodiment, the variable domain is resistant under conditions (i)and the concentration (c) is or is about 10 or 100 micrograms/mlprotease and time (t) is 1 hour. In one embodiment, the variable domainis resistant under conditions (ii) and the concentration (c′) is or isabout 40 micrograms/ml protease and time (t) is or is about 3 hours. Inone embodiment, the protease is selected from trypsin, elastase,leucozyme and pancreatin. In one embodiment, the protease is trypsin. Inone embodiment, the protease is a protease found in sputum, mucus (e.g.,gastric mucus, nasal mucus, bronchial mucus), bronchoalveolar lavage,lung homogenate, lung extract, pancreatic extract, gastric fluid, salivaor tears or the eye. In one embodiment, the protease is one found in theeye and/or tears. In one embodiment, the protease is a non-bacterialprotease. In an embodiment, the protease is an animal, eg, mammalian,eg, human, protease. In an embodiment, the protease is a GI tractprotease or a pulmonary tissue protease, eg, a GI tract protease or apulmonary tissue protease found in humans. Such protease listed here canalso be used in the methods described herein involving exposure of arepertoire of library to a protease.

In one embodiment, the variable domain is resistant to trypsin and/or atleast one other protease selected from elastase, leucozyme andpancreatin. For example, resistance is to trypsin and elastase; trypsinand leucozyme; trypsin and pacreatin; trypsin, elastase and leucozyme;trypsin, elastase and pancreatin; trypsin, elastase, pancreatin andleucozyme; or trypsin, pancreatin and leucozyme.

In one embodiment, the variable domain is displayed on bacteriophagewhen incubated under condition (i) or (ii) for example at a phagelibrary size of 10⁶ to 10¹³, eg 10⁸ to 10¹² replicative units (infectivevirions).

In one embodiment, the variable domain specifically binds VEGF followingincubation under condition (i) or (ii), eg assessed using BiaCore™ orELISA, eg phage ELISA or monoclonal phage ELISA.

In one embodiment, the variable domains of the invention specificallybind protein A or protein L. In one embodiment, specific binding toprotein A or L is present following incubation under condition (i) or(ii).

In one embodiment, the variable domains of the invention may have anOD₄₅₀ reading in ELISA, eg phage ELISA or monoclonal phage ELISA) of atleast 0.404, eg, following incubation under condition (i) or (ii).

In one embodiment, the variable domains of the invention display(substantially) a single band in gel electrophoresis, eg followingincubation under condition (i) or (ii).

In certain embodiments, the invention provides a VEGF antagonist that isa dual-specific ligand that comprises a first dAb according to theinvention that binds VEGF and a second dAb that has the same or adifferent binding specificity from the first dAb. The second dAb maybind a target selected from ApoE, Apo-SAA, BDNF, Cardiotrophin-1, CEA,CD40, CD40 Ligand, CD56, CD38, CD138, EGF, EGF receptor, ENA-78,Eotaxin, Eotaxin-2, Exodus-2, FAPa, FGF-acidic, FGF-basic, fibroblastgrowth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF,GF-131, human serum albumin, insulin, IFN-γ, IGF-I, IGF-II, IL-1α,IL-1β, IL-1 receptor, IL-1 receptor type 1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12,IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10,keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin,Mullerian inhibitory substance, monocyte colony inhibitory factor,monocyte attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1(MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-1α,MIP-1β, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor-1(MPIF-1), NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4,Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β,SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3,tumour necrosis factor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptorII, TNIL-1, TPO, VEGF, VEGF A, VEGF B, VEGF C, VEGF D, VEGF receptor 1,VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1,1-309, HER 1, HER 2, HER 3, HER 4, serum albumin, vWF, amyloid proteins(e.g., amyloid alpha), MMP12, PDK1, IgE, IL-13Rα1, IL-13Ra2, IL-15,IL-15R, IL-16, IL-17R, IL-17, IL-18, IL-18R, IL-23 IL-23R, IL-25, CD2,CD4, CD11a, CD23, CD25, CD27, CD28, CD30, CD40, CD40L, CD56, CD138,ALK5, EGFR, FcER1, TGFb, CCL2, CCL18, CEA, CR8, CTGF, CXCL12 (SDF-1),chymase, FGF, Furin, Endothelin-1, Eotaxins (e.g., Eotaxin, Eotaxin-2,Eotaxin-3), GM-CSF, ICAM-1, ICOS, IgE, IFNa, I-309, integrins,L-selectin, MIF, MIP4, MDC, MCP-1, MMPs, neutrophil elastase,osteopontin, OX-40, PARC, PD-1, RANTES, SCF, SDF-1, siglec8, TARC, TGFb,Thrombin, Tim-1, TNF, TRANCE, Tryptase, VEGF, VLA-4, VCAM, α4β7, CCR2,CCR3, CCR4, CCR5, CCR7, CCR8, alphavbeta6, alphavbeta8, cMET, CD8, vWF,amyloid proteins (e.g., amyloid alpha), MMP12, PDK1, and IgE.

In one example, the dual-specific ligand comprises a first dAb thatbinds a first epitope on VEGF and a second dAb that binds an epitope ona different target. In another example, the second dAb binds an epitopeon serum albumin.

In other embodiments, the ligand is a multispecific ligand thatcomprises a first epitope binding domain that has binding specificityfor VEGF and at least one other epitope binding domain that has bindingspecificity different from the first epitope binding domain. Forexample, the first epitope binding domain can be a dAb that binds VEGFor can be a domain that comprises the CDRs of a dAb that binds VEGF(e.g., CDRs grafted onto a suitable protein scaffold or skeleton, e.g.,an affibody, an SpA scaffold, an LDL receptor class A domain or an EGFdomain) or can be a domain that binds VEGF, wherein the domain isselected from an affibody, an SpA domain, an LDL receptor class A domainor an EGF domain).

In certain embodiments, the polypeptide, antagonist, ligand or anti-VEGFdAb monomer is characterized by one or more of the following: 1)dissociates from human VEGF with a dissociation constant (K_(d)) of 50nM to 20 pM, and a K_(off) rate constant of 5×10⁻¹ to 1×10⁻⁷ s⁻¹; asdetermined by surface plasmon resonance; 2) inhibits binding of VEGF toVEGFR2 with an IC50 of 500 nM to 50 pM; 3) neutralizes human VEGF in astandard HUVEC cell assay with an ND50 of 500 nM to 50 pM; 4)antagonizes the activity of the VEGF in a standard cell assay with anND₅₀ of ≦100 nM (5) inhibits or decreases tumour growth in a mousexenograft model; 6) resists aggregation; 7) is secreted in a quantity ofat least about 0.5 mg/L when expressed in E. coli or Pichia species(e.g., P. pastoris) or mammalian cell expression system such as CHO; 8)unfolds reversibly; or 9) has efficacy in treating, suppressing orpreventing a inflammatory disease. Reference is made to WO2006038027 andWO 2006059108 and WO 2007049017 for details of assays and tests andparameters applicable to conditions (1) to (9), and these areincorporated herein by reference.

In particular embodiments, the polypeptide, antagonist, ligand or dAbmonomer dissociates from human VEGF with a dissociation constant (K_(d))of 50 nM to 20 pM, and a K_(off) rate constant of 5×10⁻¹ to 1×10⁻⁷ s⁻¹as determined by surface plasmon resonance’; inhibits binding ofinhibits binding of VEGF to VEGFR2 (VEGF receptor 2) with an IC50 of 500nM to 50 pM; and neutralizes human VEGF in a standard HUVEC cell assaywith an ND50 of 500 nM to 50 pM. In other particular embodiments, thepolypeptide, antagonist, ligand or dAb monomer dissociates from humanVEGF with a dissociation constant (K_(d)) of 50 nM to 20 pM, and aK_(off) rate constant of 5×10⁻¹ to 1×10⁻⁷ s⁻¹ as determined by surfaceplasmon resonance; inhibits binding of VEGF to VEGFR2 with an IC50 of500 nM to 50 pM.

The protease resistant polypeptides, immunoglobulin single variabledomains and antagonists of the invention have utility in therapy,prophylaxis and diagnosis of disease or conditions in mammals, e.g.humans. In particular, they have utility as the basis of drugs that arelikely to encounter proteases when administered to a patient, such as ahuman. For example, when administered to the GI tract (eg, orally,sublingually, rectally administered), in which case the polypeptides,immunoglobulin single variable domains and antagonists may be subjectedto protease in one or more of the upper GI tract, lower GI tract, mouth,stomach, small intestine and large intestine. One embodiment, therefore,provides for a protease resistant polypeptide, immunoglobulin singlevariable domain or antagonist to be administered orally, sublingually orrectally to the GI tract of a patient to treat and/or prevent a diseaseor condition in the patient. For example, oral administration to apatient (eg, a human patient) for the treatment and/or prevention of aVEGF-mediated condition or disease such as Cancer e.g. solid tumours;inflammation and/or autoimmune disease.

In another example, the polypeptide, variable domain or antagonist islikely to encounter protease when administered (eg, by inhalation orintranasally) to pulmonary tissue (eg, the lung or airways). Oneembodiment, therefore, provides for administration of the proteaseresistant polypeptide, immunoglobulin single variable domain orantagonist to a patient (eg, to a human) by inhalation or intranasallyto pulmonary tissue of the patient to treat and/or prevent a disease orcondition in the patient. Such condition may be asthma (eg, allergicasthma), COPD, influenza or any other pulmonary disease or conditiondisclosed in WO2006038027, incorporated herein by reference. In anotherexample, the polypeptide, variable domain or antagonist is likely toencounter protease when administered (eg, by intraocular injection or aseye drops) to an eye of a patient. One embodiment, therefore, providesfor ocular administration of the protease resistant polypeptide,immunoglobulin single variable domain or antagonist to a patient (eg, toa human) by to treat and/or prevent a disease or condition (eg, adisease or condition of the eye) in the patient. Administration could betopical administration to the eye, in the form of eye drops or byinjection into the eye, eg into the vitreous humour.

One embodiment of the invention provides for a protease resistantpolypeptide, immunoglobulin single variable domain or antagonist to beadministered to the eye, e.g. in the form of eye drops or a gel or e.g.in an implant, e.g. for the treatment and/or prevention of aVEGF-mediated condition or disease of the eye such as AMD (Age relatedmacular degeneration).

In another example, the polypeptide, variable domain or antagonist islikely to encounter protease when administered (eg, by inhalation orintranasally) to pulmonary tissue (eg, the lung or airways). Oneembodiment, therefore, provides for administration to a patient (eg, toa human) by inhalation or intranasally to pulmonary tissue of thepatient to treat and/or prevent a disease or condition in the patient.Such condition may be cancer (e.g. a solid tumour, for example lung,colorectal, head and neck, pancreatic, breast, prostate, or ovariancancer), asthma (eg, allergic asthma), COPD, or any other pulmonarydisease or condition disclosed in WO2006038027, incorporated herein byreference. The antagonists, polypeptides and immunoglobulin singlevariable domains according to the invention may display improved orrelatively high melting temperatures (Tm), providing enhanced stability.High affinity target binding may also or alternatively be a feature ofthe antagonists, polypeptides and variable domains. One or more of thesefeatures, combined with protease resistance, makes the antagonists,variable domains and polypeptides amenable to use as drugs in mammals,such as humans, where proteases are particularly likely to beencountered, eg for GI tract or pulmonary tissue administration oradministration to the eye.

Thus, in one aspect, the invention provides the VEGF antagonist for oraldelivery. In one aspect, the invention provides the VEGF antagonist fordelivery to the GI tract of a patient. In one aspect, the inventionprovides the use of the VEGF antagonist in the manufacture of amedicament for oral delivery. In one aspect, the invention provides theuse of the VEGF antagonist in the manufacture of a medicament fordelivery to the GI tract of a patient. In one embodiment, the variabledomain is resistant to trypsin and/or at least one other proteaseselected from elastase, leucozyme and pancreatin. For example,resistance is to trypsin and elastase; trypsin and leucozyme; trypsinand pacreatin; trypsin, elastase and leucozyme; trypsin, elastase andpancreatin; trypsin, elastase, pancreatin and leucozyme; or trypsin,pancreatin and leucozyme.

In one aspect, the invention provides the VEGF antagonist for pulmonarydelivery. In one aspect, the invention provides the use of the VEGFantagonist in the manufacture of a medicament for pulmonary delivery. Inone aspect, the invention provides the use of the VEGF antagonist in themanufacture of a medicament for delivery to the lung of a patient. Inone embodiment, the variable domain is resistant to leucozyme.

In one aspect, the invention provides a method of oral delivery ordelivery of a medicament to the GI tract of a patient or to the lung orpulmonary tissue or eye of a patient, wherein the method comprisesadministering to the patient a pharmaceutically effective amount of aVEGF antagonist of the invention.

In one aspect, the invention provides the VEGF antagonist of theinvention for treating and/or prophylaxis of a cancer e.g. a solidtumour. In one embodiment, the solid tumour is selected from the groupconsisting of lung, colorectal, head and neck, pancreatic, breast,prostate, or ovarian cancer.

In one aspect, the invention provides the VEGF antagonist of theinvention for treating and/or prophylaxis of a vascular proliferativedisease for example angiogenesis, athersclesosis, and vascularproliferative disease in the eye such as AMD (Age Related MacularDegeneration).

In one aspect, the invention provides the VEGF antagonist of theinvention for treating and/or prophylaxis of an inflammatory condition.In one aspect, the invention provides the use of the VEGF antagonist inthe manufacture of a medicament for treating and/or prophylaxis of aninflammatory condition. In one embodiment, the condition is selectedfrom the group consisting of arthritis, multiple sclerosis, inflammatorybowel disease and chronic obstructive pulmonary disease. For example, Inone aspect, the invention provides the VEGF antagonist for treatingand/or prophylaxis of a respiratory disease. In one aspect, theinvention provides the use of the VEGF antagonist in the manufacture ofa medicament for treating and/or prophylaxis of a respiratory disease.For example, said respiratory disease is selected from the groupconsisting of lung inflammation, chronic obstructive pulmonary disease,asthma, pneumonia, hypersensitivity pneumonitis, pulmonary infiltratewith eosinophilia, environmental lung disease, pneumonia,bronchiectasis, cystic fibrosis, interstitial lung disease, primarypulmonary hypertension, pulmonary thromboembolism, disorders of thepleura, disorders of the mediastinum, disorders of the diaphragm,hypoventilation, hyperventilation, sleep apnea, acute respiratorydistress syndrome, mesothelioma, sarcoma, graft rejection, graft versushost disease, lung cancer, allergic rhinitis, allergy, asbestosis,aspergilloma, aspergillosis, bronchiectasis, chronic bronchitis,emphysema, eosinophilic pneumonia, idiopathic pulmonary fibrosis,invasive pneumococcal disease, influenza, nontuberculous mycobacteria,pleural effusion, pneumoconiosis, pneumocytosis, pneumonia, pulmonaryactinomycosis, pulmonary alveolar proteinosis, pulmonary anthrax,pulmonary edema, pulmonary embolus, pulmonary inflammation, pulmonaryhistiocytosis X, pulmonary hypertension, pulmonary nocardiosis,pulmonary tuberculosis, pulmonary veno-occlusive disease, rheumatoidlung disease, sarcoidosis, and Wegener's granulomatosis. For example,the disease is chronic obstructive pulmonary disease (COPD). Forexample, the disease is asthma.

An antagonist of the invention comprising an agent that inhibits VEGF(e.g., wherein the agent is selected from the group consisting ofantibody fragments (e.g, Fab fragment, Fab′ fragment, Fv fragment (e.g.,scFv, disulfide bonded Fv), F(ab′)₂ fragment, dAb), ligands and dAbmonomers and multimers (eg, homo- or heterodimers) can be locallyadministered to tissue or organs e.g. to pulmonary tissue (e.g., lung)or eye of a subject using any suitable method. For example, an agent canbe locally administered to pulmonary tissue via inhalation or intranasaladministration. For inhalation or intranasal administration, theantagonist of VEGF can be administered using a nebulizer, inhaler,atomizer, aerosolizer, mister, dry powder inhaler, metered dose inhaler,metered dose sprayer, metered dose mister, metered dose atomizer, orother suitable inhaler or intranasal delivery device. Thus, in oneembodiment, the invention provides a pulmonary delivery devicecontaining the VEGF antagonist. In one embodiment, the device is aninhaler or an intranasal delivery device.

In one embodiment, an agent can be locally administered to the eye viaan implantable delivery device. Thus, in one embodiment, the inventionprovides a implantable delivery device containing the VEGF antagonist

In one aspect, the invention provides an oral formulation comprising theVEGF antagonist. The formulation can be a tablet, pill, capsule, liquidor syrup. In one aspect, the invention provides an ocular formulationfor delivery to the eye comprising the VEGF antagonist e.g. theformulation can be liquid eye drops or a gel.

In one embodiment, the invention provides a pulmonary formulation fordelivery to the lung, wherein the formulation comprise an antagonist,polypeptide or variable domain of the invention with a particle sizerange of less than 5 microns, for example less than 4.5, 4, 3.5 or 3microns (eg, when in Britton-Robinson buffer, eg at a pH of 6.5 to 8.0,eg at a pH of 7 to 7.5, eg at pH7 or at pH7.5).

In one embodiment, the formulations and compositions of the inventionare provided at a pH from 6.5 to 8.0, for example 7 to 7.5, for example7, for example 7.5.

Variable domains according to any aspect of the invention may have a Tmof at least 50° C., or at least 55° C., or at least 60° C., or at least65° C., or at least 70° C. An antagonist, use, method, device orformulation of the invention may comprise such a variable domain.

In one aspect of the invention, the polypeptides, variable domains,antagonists, compositions or formulations of the invention aresubstantially stable after incubation (at a concentration of polypeptideor variable domain of 1 mg/ml) at 37 to 50° C. for 14 days in BrittonRobinson or PBS buffer. In one embodiment, at least 65, 70, 75, 80, 85,86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the polypeptide,antagonist or variable domain remains unaggregated after such incubationat 37 degrees C. In one embodiment, at least 65, 70, 75, 80, 85, 86, 87,88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the polypeptide orvariable domain remains monomeric after such incubation at 37 degrees C.In one embodiment, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98,99% of the polypeptide, antagonist or variable domain remainsunaggregated after such incubation at 50 degrees C. In one embodiment,at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of thepolypeptide or variable domain remains monomeric after such incubationat 50 degrees C. In one embodiment, no aggregation of the polypeptides,variable domains, antagonists is seen after any one of such incubations.In one embodiment, the pI of the polypeptide or variable domain remainsunchanged or substantially unchanged after incubation at 37 degrees C.at a concentration of polypeptide or variable domain of 1 mg/ml inBritton-Robinson buffer.

In one aspect of the invention, the polypeptides, variable domains,antagonists, compositions or formulations of the invention aresubstantially stable after incubation (at a concentration of polypeptideor variable domain of 100 mg/ml) at 4° C. for 7 days in Britton Robinsonbuffer or PBS at a pH of 7 to 7.5 (eg, at pH7 or pH7.5). In oneembodiment, at least 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5%of the polypeptide, antagonist or variable domain remains unaggregatedafter such incubation. In one embodiment, at least 95, 95.5, 96, 96.5,97, 97.5, 98, 98.5, 99 or 99.5% of the polypeptide or variable domainremains monomeric after such incubation. In one embodiment, noaggregation of the polypeptides, variable domains, antagonists is seenafter any one of such incubations.

In one aspect of the invention, the polypeptides, variable domains,antagonists, compositions or formulations of the invention aresubstantially stable after nebulisation (e.g. at a concentration ofpolypeptide or variable domain of 40 mg/ml) eg, at room temperature, 20degrees C. or 37° C., for 1 hour, eg jet nebuliser, eg a in a Pari LC+cup. In one embodiment, at least 65, 70, 75, 80, 85, 86, 87, 88, 90, 91,92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5% of thepolypeptide, antagonist or variable domain remains unaggregated aftersuch nebulisation. In one embodiment, at least 65, 70, 75, 80, 85, 86,87, 88, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99or 99.5% of the polypeptide or variable domain remains monomeric aftersuch nebulisation. In one embodiment, no aggregation of thepolypeptides, variable domains, antagonists is seen after any one ofsuch nebulisation.

In one aspect, the invention provides an isolated or recombinant nucleicacid encoding a polypeptide comprising an immunoglobulin single variabledomain according to any aspect of the invention or encoding apolypeptide, antagonist or variable domain according to any aspect ofthe invention. In one aspect, the invention provides a vector comprisingthe nucleic acid. In one aspect, the invention provides a host cellcomprising the nucleic acid or the vector. In one aspect, the inventionprovides a method of producing polypeptide comprising an immunoglobulinsingle variable domain, the method comprising maintaining the host cellunder conditions suitable for expression of said nucleic acid or vector,whereby a polypeptide comprising an immunoglobulin single variabledomain is produced. The method may further comprise isolating thepolypeptide, variable domain or antagonist and optionally producing avariant, eg a mutated variant, having an improved affinity and/or ND50than the isolated polypeptide variable domain or antagonist. Techniquesfor improving binding affinity of immunoglobulin single variable domainare known in the art, eg techniques for affinity maturation.

In one aspect, the invention provides a pharmaceutical compositioncomprising an immunoglobulin single variable domain, polypeptide or anantagonist of any aspect of the invention, and a pharmaceuticallyacceptable carrier, excipient or diluent.

In one embodiment, the immunoglobulin single variable domain or theantagonist of any aspect of the invention comprises an antibody constantdomain, for example, an antibody Fc, optionally wherein the N-terminusof the Fc is linked (optionally directly linked) to the C-terminus ofthe variable domain. The amino acid sequence of a suitable Fc is shownin FIG. 52 b.

The polypeptide or variable domain of the invention can be isolatedand/or recombinant.

In one aspect, the invention is a method for selecting a proteaseresistant peptide or polypeptide, for example an antagonist of vascularendothelial growth factor (VEGF), e.g. an anti-VEGF dAb. The methodcomprises providing a repertoire of peptides or polypeptides, combiningthe repertoire and a protease under conditions suitable for proteaseactivity, and recovering a peptide or polypeptide that has a desiredbiological activity, whereby a protease resistant peptide or polypeptideis selected.

The repertoire and the protease are generally incubated for a period ofat least about 30 minutes. Any desired protease can be used in themethod, such as one or more of the following, serine protease, cysteineprotease, aspartate proteases, thiol proteases, matrix metalloprotease,carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B),trypsin, chymotrypsin, pepsin, papain, elastase, leukozyme, pancreatin,thrombin, plasmin, cathepsins (e.g., cathepsin G), proteinase (e.g.,proteinase 1, proteinase 2, proteinase 3), thermolysin, chymosin,enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4, caspase5, caspase 9, caspase 12, caspase 13), calpain, ficain, clostripain,actinidain, bromelain, and separase. In particular embodiments, theprotease is trypsin, elastase or leucozyme. The protease can also beprovided by a biological extract, biological homogenate or biologicalpreparation. If desired, the method further comprises adding a proteaseinhibitor to the combination of the repertoire and the protease afterincubation is complete.

In some embodiments, a peptide or polypeptide that has a desiredbiological activity is recovered based on a binding activity. Forexample, the peptide or polypeptide can be recovered based on binding ageneric ligand, such as protein A, protein G or protein L. The bindingactivity can also be specific binding to a target ligand. Exemplarytarget ligands include ApoE, Apo-SAA, BDNF, Cardiotrophin-1, CEA, CD40,CD40 Ligand, CD56, CD38, CD138, EGF, EGF receptor, ENA-78, Eotaxin,Eotaxin-2, Exodus-2, FAPa, FGF-acidic, FGF-basic, fibroblast growthfactor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-β1,human serum albumin, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β, IL-1receptor, IL-1 receptor type 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8(72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10, keratinocytegrowth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerianinhibitory substance, monocyte colony inhibitory factor, monocyteattractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF),MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-1α, MIP-1β,MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M,PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDFβ3, SCF, SCGF, stemcell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumour necrosisfactor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1,TPO, VEGF, VEGF A, VEGF B, VEGF C, VEGF D, VEGF receptor 1, VEGFreceptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309,HER 1, HER 2, HER 3, HER 4, serum albumin, vWF, amyloid proteins (e.g.,amyloid alpha), MMP12, PDK1, IgE, IL-13Rα1, IL-13Rα2, IL-15, IL-15R,IL-16, IL-17R, IL-17, IL-18, IL-18R, IL-23 IL-23R, IL-25, CD2, CD4,CD11a, CD23, CD25, CD27, CD28, CD30, CD40, CD40L, CD56, CD138, ALK5,EGFR, FcER1, TGFb, CCL2, CCL18, CEA, CR8, CTGF, CXCL12 (SDF-1), chymase,FGF, Furin, Endothelin-1, Eotaxins (e.g., Eotaxin, Eotaxin-2,Eotaxin-3), GM-CSF, ICAM-1, ICOS, IgE, IFNa, 1-309, integrins,L-selectin, MIF, MIP4, MDC, MCP-1, MMPs, neutrophil elastase,osteopontin, OX-40, PARC, PD-1, RANTES, SCF, SDF-1, siglec8, TARC, TGFb,Thrombin, Tim-1, TNF, TRANCE, Tryptase, VEGF, VLA-4, VCAM, α4β7, CCR2,CCR3, CCR4, CCR5, CCR7, CCR8, alphavbeta6, alphavbeta8, cMET, CD8, vWF,amyloid proteins (e.g., amyloid alpha), MMP12, PDK1, and IgE.

In particular embodiments, the peptide or polypeptide is recovered bypanning.

In some embodiments, the repertoire comprises a display system. Forexample, the display system can be bacteriophage display, ribosomedisplay, emulsion compartmentalization and display, yeast display,puromycin display, bacterial display, display on plasmid, or covalentdisplay. Exemplary display systems link coding function of a nucleicacid and functional characteristics of the peptide or polypeptideencoded by the nucleic acid. In particular embodiments, the displaysystem comprises replicable genetic packages.

In some embodiments, the display system comprises bacteriophage display.For example, the bacteriophage can be fd, M13, lambda, MS2 or T7. Inparticular embodiments, the bacteriophage display system is multivalent.In some embodiments, the peptide or polypeptide is displayed as a pIIIfusion protein.

In other embodiments, the method further comprises amplifying thenucleic acid encoding a peptide or polypeptide that has a desiredbiological activity. In particular embodiments, the nucleic acid isamplified by phage amplification, cell growth or polymerase chainreaction.

In some embodiments, the repertoire is a repertoire of immunoglobulinsingle variable domains, which for example are bind to and areantagonists of vascular endothelial growth factor (VEGF). In particularembodiments, the immunoglobulin single variable domain is a heavy chainvariable domain. In more particular embodiments, the heavy chainvariable domain is a human heavy chain variable domain. In otherembodiments, the immunoglobulin single variable domain is a light chainvariable domain. In particular embodiments, the light chain variabledomain is a human light chain variable domain.

In another aspect, the invention is a method for selecting a peptide orpolypeptide that binds a target ligand e.g. VEGF, with high affinityfrom a repertoire of peptides or polypeptides. The method comprisesproviding a repertoire of peptides or polypeptides, combining therepertoire and a protease under conditions suitable for proteaseactivity, and recovering a peptide or polypeptide that binds the targetligand.

The repertoire and the protease are generally incubated for a period ofat least about 30 minutes. Any desired protease can be used in themethod, such as one or more of the following, serine protease, cysteineprotease, aspartate proteases, thiol proteases, matrix metalloprotease,carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B),trypsin, chymotrypsin, pepsin, papain, elastase, leukozyme, pancreatin,thrombin, plasmin, cathepsins (e.g., cathepsin G), proteinase (e.g.,proteinase 1, proteinase 2, proteinase 3), thermolysin, chymosin,enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4, caspase5, caspase 9, caspase 12, caspase 13), calpain, ficain, clostripain,actinidain, bromelain, and separase. In particular embodiments, theprotease is trypsin, elastase or leucozyme. The protease can also beprovided by a biological extract, biological homogenate or biologicalpreparation. If desired, the method further comprises adding a proteaseinhibitor to the combination of the repertoire and the protease afterincubation is complete.

The peptide or polypeptide can be recovered based on binding any desiredtarget ligand, such as the target ligands disclosed herein. Inparticular embodiments, the peptide or polypeptide is recovered bypanning.

In some embodiments, the repertoire comprises a display system. Forexample, the display system can be bacteriophage display, ribosomedisplay, emulsion compartmentalization and display, yeast display,puromycin display, bacterial display, display on plasmid, or covalentdisplay. Exemplary display systems link coding function of a nucleicacid and functional characteristics of the peptide or polypeptideencoded by the nucleic acid. In particular embodiments, the displaysystem comprises replicable genetic packages.

In some embodiments, the display system comprises bacteriophage display.For example, the bacteriophage can be fd, M13, lambda, MS2 or T7. Inparticular embodiments, the bacteriophage display system is multivalent.In some embodiments, the peptide or polypeptide is displayed as a pIIIfusion protein.

In other embodiments, the method further comprises amplifying thenucleic acid encoding a peptide or polypeptide that has a desiredbiological activity. In particular embodiments, the nucleic acid isamplified by phage amplification, cell growth or polymerase chainreaction.

In some embodiments, the repertoire is a repertoire of immunoglobulinsingle variable domains, e.g. which bind to and are antagonists of VEGF.In particular embodiments, the immunoglobulin single variable domain isa heavy chain variable domain. In more particular embodiments, the heavychain variable domain is a human heavy chain variable domain. In otherembodiments, the immunoglobulin single variable domain is a light chainvariable domain. In particular embodiments, the light chain variabledomain is a human light chain variable domain.

In another aspect, the invention is a method of producing a repertoireof protease resistant peptides or polypeptides. The method comprisesproviding a repertoire of peptides or polypeptides, combining therepertoire of peptides or polypeptides and a protease under suitableconditions for protease activity, and recovering a plurality of peptidesor polypeptides that have a desired biological activity, whereby arepertoire of protease resistant peptides or polypeptides is produced.

In some embodiments, the repertoire and the protease are incubated for aperiod of at least about 30 minutes. For example, the protease used inthe method can be one or more of the following, serine protease,cysteine protease, aspartate proteases, thiol proteases, matrixmetalloprotease, carboxypeptidase (e.g., carboxypeptidase A,carboxypeptidase B), trypsin, chymotrypsin, pepsin, papain, elastase,leukozyme, pancreatin, thrombin, plasmin, cathepsins (e.g., cathepsinG), proteinase (e.g., proteinase 1, proteinase 2, proteinase 3),thermolysin, chymosin, enteropeptidase, caspase (e.g., caspase 1,caspase 2, caspase 4, caspase 5, caspase 9, caspase 12, caspase 13),calpain, ficain, clostripain, actinidain, bromelain, and separase. Inparticular embodiments, the protease is trypsin, elastase or leucozyme.The protease can also be provided by a biological extract, biologicalhomogenate or biological preparation. If desired, the method furthercomprises adding a protease inhibitor to the combination of therepertoire and the protease after incubation is complete.

In some embodiments, a plurality of peptides or polypeptides that have adesired biological activity is recovered based on a binding activity.For example, a plurality of peptides or polypeptides can be recoveredbased on binding a generic ligand, such as protein A, protein G orprotein L. The binding activity can also be specific binding to a targetligand, such as a target ligand described herein. In particularembodiments, a plurality of peptides or polypeptides that has thedesired biological activity is recovered by panning.

In some embodiments, the repertoire comprises a display system. Forexample, the display system can be bacteriophage display, ribosomedisplay, emulsion compartmentalization and display, yeast display,puromycin display, bacterial display, display on plasmid, or covalentdisplay. In particular embodiments, the display system links codingfunction of a nucleic acid and functional characteristics of the peptideor polypeptide encoded by the nucleic acid. In particular embodiments,the display system comprises replicable genetic packages.

In some embodiments, the display system comprises bacteriophage display.For example, the bacteriophage can be fd, M13, lambda, MS2 or T7. Inparticular embodiments, the bacteriophage display system is multivalent.In some embodiments, the peptide or polypeptide is displayed as a pIIIfusion protein.

In other embodiments, the method further comprises amplifying thenucleic acids encoding a plurality of peptides or polypeptides that havea desired biological activity. In particular embodiments, the nucleicacids are amplified by phage amplification, cell growth or polymerasechain reaction.

In some embodiments, the repertoire is a repertoire of immunoglobulinsingle variable domains, e.g. which bind to and are antagonists of VEGF.In particular embodiments, the immunoglobulin single variable domain isa heavy chain variable domain. In more particular embodiments, the heavychain variable domain is a human heavy chain variable domain. In otherembodiments, the immunoglobulin single variable domain is a light chainvariable domain. In particular embodiments, the light chain variabledomain is a human light chain variable domain.

In another aspect, the invention is a method for selecting a proteaseresistant polypeptide comprising an immunoglobulin single variabledomain (dAb) that binds a target ligand, e.g. VEGF from a repertoire. Inone embodiment, the method comprises providing a phage display systemcomprising a repertoire of polypeptides that comprise an immunoglobulinsingle variable domain, combining the phage display system and aprotease selected from the group consisting of elastase, leucozyme andtrypsin, under conditions suitable for protease activity, and recoveringa phage that displays a polypeptide comprising an immunoglobulin singlevariable domain that binds the target ligand.

In some embodiments, the protease is used at 100 μg/ml, and the combinedphage display system and protease are incubated at about 37° C.overnight.

In some embodiments, the phage that displays a polypeptide comprising animmunoglobulin single variable domain that binds the target ligand isrecovered by binding to said target. In other embodiments, the phagethat displays a polypeptide comprising an immunoglobulin single variabledomain that binds the target ligand is recovered by panning.

The invention also relates to an isolated protease resistant peptide orpolypeptide selectable or selected by the methods described herein. In aparticular embodiment, the invention relates to an isolated protease(e.g., trypsin, elastase, leucozyme) resistant immunoglobulin singlevariable domain (e.g., human antibody heavy chain variable domain, humanantibody light chain variable domain) selectable or selected by themethods described herein.

The invention also relates to an isolated or recombinant nucleic acidthat encodes a protease resistant peptide or polypeptide (e.g.,trypsin-, elastase-, or leucozyme-resistant immunoglobulin singlevariable domain) selectable or selected by the methods described herein,and to vectors (e.g., expression vectors) and host cells that comprisethe nucleic acids.

The invention also relates to a method for making a protease resistantpeptide or polypeptide (e.g., trypsin-, elastase-, orleucozyme-resistant immunoglobulin single variable domain) selectable orselected by the methods described herein, comprising maintaining a hostcell that contains a recombinant nucleic acid encoding the proteaseresistant peptide or polypeptide under conditions suitable forexpression, whereby a protease resistant peptide or polypeptide isproduced.

The invention also relates to a protease resistant peptide orpolypeptide (e.g., trypsin-, elastase-, or leucozyme-resistantimmunoglobulin single variable domain) selectable or selected by themethods described herein for use in medicine (e.g., for therapy ordiagnosis). The invention also relates to use of a protease resistantpeptide or polypeptide (e.g., trypsin-, elastase-, orleucozyme-resistant immunoglobulin single variable domain) selectable orselected by the methods described herein for the manufacture of amedicament for treating disease. The invention also relates to a methodof treating a disease, comprising administering to a subject in needthereof, an effective amount of a protease resistant peptide orpolypeptide (e.g., trypsin-, elastase-, or leucozyme-resistantimmunoglobulin single variable domain) selectable or selected by themethods described herein.

The invention also relates to a diagnostic kit for determine whetherVEGF is present in a sample or how much VEGF is present in a sample,comprising a polypeptide, immunoglobulin variable domain (dAb), orantagonist of the invention and instructions for use (e.g., to determinethe presence and/or quantity of VEGF in the sample). In someembodiments, the kit further comprises one or more ancillary reagents,such as a suitable buffer or suitable detecting reagent (e.g., adetectably labeled antibody or antigen-binding fragment thereof thatbinds the polypeptide or dAb of the invention or a moiety associated orconjugated thereto.

The invention also relates to a device comprising a solid surface onwhich a polypeptide antagonist or dAb of the invention is immobilizedsuch that the immobilized polypeptide or dAb binds VEGF. Any suitablesolid surfaces on which an antibody or antigen-binding fragment thereofcan be immobilized can be used, for example, glass, plastics,carbohydrates (e.g., agarose beads). If desired the support can containor be modified to contain desired functional groups to facilitateimmobilization. The device, and or support, can have any suitable shape,for example, a sheet, rod, strip, plate, slide, bead, pellet, disk, gel,tube, sphere, chip, plate or dish, and the like. In some embodiments,the device is a dipstick.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the multiple cloning site of pDOM13 (akapDOM33), which was used to prepare a phage display repertoire.

FIG. 2 shows several Novex 10-20% Tricene gels run with samples fromdifferent time points of dAbs that were incubated with trypsin at 40ug/ml at 30° C. Samples were taken immediately before the addition oftrypsin, and then at one hour, three hours and 24 hours after theaddition of trypsin. The proteins were stained with 1× SureBlue. Thegels illustrate that both DOM15-10 and DOM15-26-501 were significantlydigested during the first three hours of incubation with trypsin.Digestion of DOM15-26, DOM4-130-54 and DOM1h-131-511 only becameapparent after 24 hours of incubation with trypsin.

FIG. 3 is an illustration of the amino acid sequences of DOM1h-131-511and 24 selected variants. The amino acids that differ from the parentsequence in selected clones are highlighted (those that are identicalare marked by dots). The loops corresponding to CDR1, CDR2 and CDR3 areoutlined with boxes.

FIG. 4 is an illustration of the amino acid sequences of DOM4-130-54 and27 selected variants. The amino acids that differ from the parentsequence in selected clones are highlighted (those that are identicalare marked by dots). The loops corresponding to CDR1, CDR2 and CDR3 areoutlined with boxes.

FIG. 5 is an illustration of the amino acid sequence of DOM15-26-555 and21 selected variants. The amino acids that differ from the parentsequence in selected clones are highlighted (those that are identicalare marked by dots). The loops corresponding to CDR1, CDR2 and CDR3 areoutlined with boxes.

FIG. 6 is an illustration of the amino acid sequence of DOM15-10 and 16selected variants. The amino acids that differ from the parent sequencein selected clones are highlighted (those that are identical are markedby dots). The loops corresponding to CDR1, CDR2 and CDR3 are outlinedwith boxes.

FIGS. 7A-7D are BIAcore traces showing bind of a parent dAb,DOM1h-131-511 (FIG. 7A) and three variant dAbs, DOM1h-131-203 (FIG. 7B),DOM1h-131-204 (FIG. 7C) and DOM1h-131-206 (FIG. 7D), to immoblized TNFR1after incubation with different concentrations of trypsin (ranging from0 to 100 μg/ml) overnight at 37° C. The results show that all threevariants are more resistant than the parent to proteolysis at highconcentrations of trypsin (100 ug/ml).

FIGS. 8A-8C are BIAcore traces showing binding of dAbs DOM1h-131-511(FIG. 8A), DOM1h-131-202 (FIG. 8B) and DOM1h-131-206 (FIG. 8C) toimmobilized TNFR1 after incubation with elastase and leucozymeovernight. The dAbs showed increased resistance to proteolysis comparedto the parent against both elastase and leucozyme.

FIG. 9 shows two 4-12% Novex Bis-Tris gels run with samples of dAbsDOM1h-131-511, DOM1h-131-203, DOM1h-131-204, DOM1h-131-206,DOM1h-131-54, DOM1h-131-201, and DOM1h-131-202 before incubation withtrypsin and samples after incubation with 100 μg/ml of trypsin for 1hour, 3 hours and 24 hours.

FIGS. 10A-10C are BIAcore traces showing binding of DOM4-130-54 (FIG.10A), DOM4-130-201 (FIG. 10B) and DOM4-130-202 (FIG. 10C) to immobilizedIL-1R1 fusion protein after incubation with different concentrations oftrypsin (ranging from 0 to 100 μg/ml) overnight at 37° C. The resultsshow that both variants are more resistant than their parent toproteolysis at high concentrations of trypsin (100 μg/ml).

FIGS. 11A-11C are BIAcore traces showing binding of DOM4-130-54 (FIG.11A), DOM4-130-201 (FIG. 11B) and DOM4-130-202 (FIG. 11C) to immobilizedIL-1R1 fusion protein after incubation with elastase and leucozymeovernight. The dAbs showed increased resistance to proteolysis comparedto parent against both proteases tested.

FIG. 12 is an illustration of the amino acid sequence of DOM15-26-555and 6 variants. The amino acids that differ from the parent sequence inselected clones are highlighted (those that are identical are marked bydots).

FIGS. 13A and 13B are BIAcore traces showing binding of the parent dAb,DOM15-26-555 (FIG. 13A) and the most protease resistant variant,DOM15-26-593 (FIG. 13B) to immobilized VEGF. The parent and the variantwere compared on the BIAcore for hVEGF binding at the dAb concentrationof 100 nM after incubation with trypsin at a concentration of 200 μg/ml.The reaction was carried out for three hours or 24 hours at 37° C. Theresults show that the variant is more resistant than the parent toproteolysis after 24 hours of trypsin treatment.

FIG. 14 is a graph showing effects of trypsin treatment on hVEGF bindingby DOM15-26-555 variants. The results clearly show that all variants aremore resistant than the parent (DOM15-26-555) to proteolysis after 24hours of trypsin treatment.

FIG. 15 shows two Novex 10-20% Tricine gels that were loaded with 15 μgof treated and untreated samples of DOM15-26-555 or DOM15-26-593.Samples were taken immediately before the addition of trypsin, and thenat one hour, three hours and 24 hours after the addition of trypsin. Theproteins were stained with 1× SureBlue. The gels illustrate that thetrypsin resistance profile of DOM15-26-593 varied from the profile shownby the BIAcore experiment.

FIG. 16 is an illustration of the amino acid sequence of DOM15-10 and avariant, DOM15-10-11. The amino acids that differ from the parentsequence in the variant are highlighted (those that are identical aremarked by dots).

FIGS. 17A and 17B are BIAcore traces showing binding of the parent,DOM15-10 (FIG. 17A) and the variant, DOM15-10-11 (FIG. 17B), toimmobilized VEGF. The parent and the variant were compared on theBIAcore for hVEGF binding at the dAb concentration of 100 nM afterincubation with trypsin at a concentration of 200 μg/ml. The reactionwas carried out for one hour, three hours and 24 hours at 37° C. Theresults show that the variant is more resistant than the parent toproteolysis after 24 hours of trypsin treatment.

FIG. 18 shows two Novex 10-20% Tricene gels that were loaded with 15 μgof samples of DOM15-10 and DOM15-10-11. Samples were taken immediatelybefore the addition of trypsin, and then at one hour, three hours, and24 hours after the addition of trypsin. The proteins were stained withSureBlue (1×). The results show that the binding activity seen in theBIAcore study directly reflects the protein's integrity.

FIGS. 19A-19L illustrate the nucleotide sequences of several nucleicacids encoding dAbs that are variants of DOM1h-131-511 or DOM4-130-54.The nucleotide sequences encode the amino acid sequences presented inFIG. 3 and FIG. 4, respectively.

FIGS. 20A-20E illustrate the nucleotide sequences of several nucleicacids encoding dAbs that are variants of DOM15-26-555 or DOM15-10. Thenucleotide sequences encode the amino acid sequences presented in FIG. 5and FIG. 6, respectively.

FIG. 21 shows a vector map of pDOM 38.

FIG. 22: Shows a Gel run on Labchip of DOM10-53-474 and DOM15-26-593proteins treated with trypsin at 25:1 dAb:trypsin ratio at 30° C. fordifferent time points. Arrows show full length protein.

FIG. 23: Is a Size exclusion chromatography trace showing the high levelof purity obtained for each sample after purification by MMCchromatography followed by anion exchange. The UV was monitored at 225nm and the column was run in 1×PBS with 10% ethanol (v/v). Thepercentage monomer was calculated by integration of the peak area withbaseline correction.

FIG. 24: Shows Protease stability data for DOM1h-131-511, DOM1h-131-202and DOM1h-131-206.

FIG. 25: Is an SEC which illustrates 14 day stability data ofDOM1h-131-202, DOM1h-131-206 and DOM1h-131-511 in Britton-Robinsonbuffer at 37 and 50° C. The protein concentration for all the dAbs was 1mg/ml. SEC was used to determine if any changes had occurred in theprotein during thermal stress and the amount of monomer left in solutionrelative to the time=0 (T0) sample.

FIGS. 26 A to I: Show SEC traces showing the effect of thermal stress(37 and 50° C.) on DOM1h-131-511 (A to C), -202 (D to F) and -206 (G toI). Also shown is the percentage of monomer left in solution relative tothe T=0 at the given time point.

FIG. 27: Shows IEF analysis of DOM1h-131-202, DOM1h-131-206 andDOM1h-131-511 at 24 hr, 48 hr and 7 and 14 days thermal stress. Thesamples had been incubated at either 37 or 50° C. in Britton-Robinsonbuffer.

FIG. 28: TNFR-1 RBA showing the effect of 14 days incubation ofDOM1h-131-202, DOM1h-131-206 and DOM1h-131-511 at 50° C. The proteinconcentration was assumed to be 1 mg/ml. A negative control dAb (VHdummy) which does not bind antigen is also shown.

FIG. 29: Illustrates Effects of storing A: DOM1h-131-202, B:DOM1h-131-206 and C: DOM1h-131-511 at ˜100 mg/ml for 7 days inBritton-Robinson buffer at +4° C. The UV was monitored at 280 nm.

FIG. 30: Shows data from Nebuliser testing of DOM1h-131-202,DOM1h-131-206 and DOM1h-131-511 in the Pari E-flow and LC+. The proteinconcentration was 5 mg/ml in either Britton-Robinson buffer.

FIG. 31: Illustrates the Relative percentage changes in monomerconcentrations during nebulisation of DOM1h-131-202, DOM1h-131-206 andDOM1h-131-511 in Britton-Robinson buffer at 5 mg/ml.

FIG. 32: Shows SEC traces of DOM1h-131-206 and DOM1h-131-511 inBritton-Robinson buffer post nebulisation from the Pari LC+.

FIG. 33: Shows SEC traces of DOM1h-131-206 during the nebulisationprocess over 1 hour at 40 mg/ml in PBS. The protein in both thenebuliser cup and aerosol are highly resistance to the effects of shearand thermal stress that may be experienced by the dAb duringnebulisation.

FIG. 34: Shows the sedimentation velocity curves for each of the threelead proteins (DOM1h-131-206 and DOM1h-131-511 and DOM1h-131-202) Thebimodal peak observed for the lower concentration sample ofDOM1h-131-206 is an artefact owing to a sample leak from the cell inthis instance.

FIG. 35: Shows the effect of buffer and device on nebulised droplet sizeof GSK 1995056A (DOM1h-131-511).

FIG. 36: Stability of GSK1995056A (DOM1h-131-511) after nebulisation invarious devices assessed by dimer formation as measured by SEC.

FIG. 37: Shows Nebuliser testing of GSK1922567A (202), GSK1995057A (206)and GSK1995056A (511) in the Pari E-flow and LC+. A) testing inBritton-Robinson buffer, B) testing in PEG1000/sucrose buffer.

FIG. 38: Depicts a TNF-α dose curve in the human TNFR1 receptor bindingassay. Each sample was tested as four replicates.

FIG. 39: Shows Inhibition by GSK1922567A(DOM1h-131-202), GSK1995057A(DOM1h-131-206) and GSK1995056A (DOM1h-131-511) in the human TNFR1receptor binding assay. Each sample was tested as four replicates.

FIG. 40: Illustrates potency of the DOM15-26 and DOM15-26-593 dAbs inthe VEGF RBA.

FIG. 41: Shows pharmacokinetics of DMS1529 (DOM15-26-593) and DMS1545(DOM15-26-501) after single bolus dose i.v. administration to rats at 5mg/mg

FIG. 42 a: Shows SEC-MALLs (Size exclusion chromatograph-multi-anglelaser light scattering) analysis of DMS1529 Fc fusion (DOM15-26-593 Fcfusion) confirming monomeric properties. Two different batches are shownthat demonstrate similar properties with regard to refractive index(i.e. concentration; broken lines) and light scattering (solid lines).The line marked with the arrow signifies the molecular mass calculation.

FIG. 42 b: Shows AUC (analytical ultracentrifugation) analysis ofDMS1529 Fc fusion (DOM15-26-593 Fc fusion) confirming monomericproperties. One batch of material was tested at three differentconcentrations, approximating to 0.2, 0.5 & 1.0 mg/ml in PBS buffer. Theanalysis of the sedimentation rate confirmed a molecular mass of approx.80 kDa.

FIG. 43: Shows DSC traces of DMS1529 (DOM15-26-593) and DOM15-26-501.

FIG. 44: Is a VEGF Binding ELISA for DMS1529 (DOM15-26-593) before andafter, 10 freeze-thaw cycles on two different batches of material.

FIG. 45: Shows the consistency of DOM15-26-593 SEC profile before andafter 10 freeze thaw cycles.

FIG. 46: Illustrates results from an accelerated stability study of theDMS1529 fusion (DOM15-26-593 Fc fusion); binding ELISA demonstratingactivity after 7 days incubation at the temperature shown.

FIG. 47A: Shows stability of DMS1529 (DOM15-26-593) in human cynomolgusafter 14 & 15 days incubation at 37° C.

FIG. 47B: Shows stability of DMS1529 (DOM15-26-593) in human serum after14 & 15 days incubation at 37° C.

FIG. 48: Shows potency of DOM15-26 & DOM15-26-593 dAbs as Fc fusions(DMS1564 & 1529 respectively) in the VEGF RBA.

FIG. 49: Illustrates inhibition of HUVEC cell proliferation by theDMS1529 fusion (DOM15-26-593 FC fusion).

FIG. 50: pDom33 vector map.

FIG. 51 a: Depicts amino acid sequences of dAbs that bind serum albumin.

FIG. 51 b: Depicts nucleic acid sequences of dAbs that bind serumalbumin.

FIG. 52 a: Depicts the amino acid sequence of DOM15-26-593-Fc fusion

FIG. 52 b: Depicts the amino acid sequence of an antibody Fc

FIG. 52 c: Depicts the nucleic acid sequence of DOM15-26-593-Fc fusion.

DETAILED DESCRIPTION OF THE INVENTION

Within this specification the invention has been described, withreference to embodiments, in a way which enables a clear and concisespecification to be written. It is intended and should be appreciatedthat embodiments may be variously combined or separated without partingfrom the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)Ed, John Wiley & Sons, Inc. which are incorporated herein by reference)and chemical methods.

As used herein, the term “antagonist of vascular endothelial growthfactor (VEGF)” or “anti-VEGF antagonist” or the like refers to an agent(e.g., a molecule, a compound) which binds VEGF and can inhibit a (i.e.,one or more) function of VEGF.

As used herein, “peptide” refers to about two to about 50 amino acidsthat are joined together via peptide bonds.

As used herein, “polypeptide” refers to at least about 50 amino acidsthat are joined together by peptide bonds. Polypeptides generallycomprise tertiary structure and fold into functional domains.

As used herein, a peptide or polypeptide (e.g. a domain antibody (dAb))that is “resistant to protease degradation” is not substantiallydegraded by a protease when incubated with the protease under conditionssuitable for protease activity. A polypeptide (e.g., a dAb) is notsubstantially degraded when no more than about 25%, no more than about20%, no more than about 15%, no more than about 14%, no more than about13%, no more than about 12%, no more than about 11%, no more than about10%, no more than about 9%, no more than about 8%, no more than about7%, no more than about 6%, no more than about 5%, no more than about 4%,no more than about 3%, no more that about 2%, no more than about 1%, orsubstantially none of the protein is degraded by protease afterincubation with the protease for about one hour at a temperaturesuitable for protease activity. For example at 37 or 50 degrees C.Protein degradation can be assessed using any suitable method, forexample, by SDS-PAGE or by functional assay (e.g., ligand binding) asdescribed herein.

As used herein, “display system” refers to a system in which acollection of polypeptides or peptides are accessible for selectionbased upon a desired characteristic, such as a physical, chemical orfunctional characteristic. The display system can be a suitablerepertoire of polypeptides or peptides (e.g., in a solution, immobilizedon a suitable support). The display system can also be a system thatemploys a cellular expression system (e.g., expression of a library ofnucleic acids in, e.g., transformed, infected, transfected or transducedcells and display of the encoded polypeptides on the surface of thecells) or an acellular expression system (e.g., emulsioncompartmentalization and display). Exemplary display systems link thecoding function of a nucleic acid and physical, chemical and/orfunctional characteristics of a polypeptide or peptide encoded by thenucleic acid. When such a display system is employed, polypeptides orpeptides that have a desired physical, chemical and/or functionalcharacteristic can be selected and a nucleic acid encoding the selectedpolypeptide or peptide can be readily isolated or recovered. A number ofdisplay systems that link the coding function of a nucleic acid andphysical, chemical and/or functional characteristics of a polypeptide orpeptide are known in the art, for example, bacteriophage display (phagedisplay, for example phagemid display), ribosome display, emulsioncompartmentalization and display, yeast display, puromycin display,bacterial display, display on plasmid, covalent display and the like.(See, e.g., EP 0436597 (Dyax), U.S. Pat. No. 6,172,197 (McCafferty etal.), U.S. Pat. No. 6,489,103 (Griffiths et al.).)

As used herein, “repertoire” refers to a collection of polypeptides orpeptides that are characterized by amino acid sequence diversity. Theindividual members of a repertoire can have common features, such ascommon structural features (e.g., a common core structure) and/or commonfunctional features (e.g., capacity to bind a common ligand (e.g., ageneric ligand or a target ligand)).

As used herein, “functional” describes a polypeptide or peptide that hasbiological activity, such as specific binding activity. For example, theterm “functional polypeptide” includes an antibody or antigen-bindingfragment thereof that binds a target antigen through its antigen-bindingsite.

As used herein, “generic ligand” refers to a ligand that binds asubstantial portion (e.g., substantially all) of the functional membersof a given repertoire. A generic ligand (e.g., a common generic ligand)can bind many members of a given repertoire even though the members maynot have binding specificity for a common target ligand. In general, thepresence of a functional generic ligand-binding site on a polypeptide(as indicated by the ability to bind a generic ligand) indicates thatthe polypeptide is correctly folded and functional. Suitable examples ofgeneric ligands include superantigens, antibodies that bind an epitopeexpressed on a substantial portion of functional members of arepertoire, and the like.

“Superantigen” is a term of art that refers to generic ligands thatinteract with members of the immunoglobulin superfamily at a site thatis distinct from the target ligand-binding sites of these proteins.Staphylococcal enterotoxins are examples of superantigens which interactwith T-cell receptors. Superantigens that bind antibodies includeProtein G, which binds the IgG constant region (Bjorck and Kronvall, J.Immunol., 133:969 (1984)); Protein A which binds the IgG constant regionand V_(H) domains (Forsgren and Sjoquist, J. Immunol., 97:822 (1966));and Protein L which binds V_(L) domains (Bjorck, J. Immunol., 140:1194(1988)).

As used herein, “target ligand” refers to a ligand which is specificallyor selectively bound by a polypeptide or peptide. For example, when apolypeptide is an antibody or antigen-binding fragment thereof, thetarget ligand can be any desired antigen or epitope. Binding to thetarget antigen is dependent upon the polypeptide or peptide beingfunctional.

As used herein an antibody refers to IgG, IgM, IgA, IgD or IgE or afragment (such as a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, closedconformation multispecific antibody, disulphide-linked scFv, diabody)whether derived from any species naturally producing an antibody, orcreated by recombinant DNA technology; whether isolated from serum,B-cells, hybridomas, transfectomas, yeast or bacteria.

As used herein, “antibody format” refers to any suitable polypeptidestructure in which one or more antibody variable domains can beincorporated so as to confer binding specificity for antigen on thestructure. A variety of suitable antibody formats are known in the art,such as, chimeric antibodies, humanized antibodies, human antibodies,single chain antibodies, bispecific antibodies, antibody heavy chains,antibody light chains, homodimers and heterodimers of antibody heavychains and/or light chains, antigen-binding fragments of any of theforegoing (e.g., a Fv fragment (e.g., single chain Fv (scFv), adisulfide bonded Fv), a Fab fragment, a Fab′ fragment, a F(ab′)₂fragment), a single antibody variable domain (e.g., a dAb, V_(H),V_(HH), V_(L)), and modified versions of any of the foregoing (e.g.,modified by the covalent attachment of polyethylene glycol or othersuitable polymer or a humanized V_(HH)).

The phrase “immunoglobulin single variable domain” refers to an antibodyvariable domain (V_(H), V_(HH), V_(L)) that specifically binds anantigen or epitope independently of other V regions or domains. Animmunoglobulin single variable domain can be present in a format (e.g.,homo- or hetero-multimer) with other variable regions or variabledomains where the other regions or domains are not required for antigenbinding by the single immunoglobulin variable domain (i.e., where theimmunoglobulin single variable domain binds antigen independently of theadditional variable domains). A “domain antibody” or “dAb” is the sameas an “immunoglobulin single variable domain” as the term is usedherein. A “single immunoglobulin variable domain” is the same as an“immunoglobulin single variable domain” as the term is used herein. A“single antibody variable domain” is the same as an “immunoglobulinsingle variable domain” as the term is used herein. An immunoglobulinsingle variable domain is in one embodiment a human antibody variabledomain, but also includes single antibody variable domains from otherspecies such as rodent (for example, as disclosed in WO 00/29004, thecontents of which are incorporated herein by reference in theirentirety), nurse shark and Camelid V_(HH) dAbs. Camelid V_(HH) areimmunoglobulin single variable domain polypeptides that are derived fromspecies including camel, llama, alpaca, dromedary, and guanaco, whichproduce heavy chain antibodies naturally devoid of light chains. TheV_(HH) may be humanized.

A “domain” is a folded protein structure which has tertiary structureindependent of the rest of the protein. Generally, domains areresponsible for discrete functional properties of proteins, and in manycases may be added, removed or transferred to other proteins withoutloss of function of the remainder of the protein and/or of the domain. A“single antibody variable domain” is a folded polypeptide domaincomprising sequences characteristic of antibody variable domains. Ittherefore includes complete antibody variable domains and modifiedvariable domains, for example, in which one or more loops have beenreplaced by sequences which are not characteristic of antibody variabledomains, or antibody variable domains which have been truncated orcomprise N- or C-terminal extensions, as well as folded fragments ofvariable domains which retain at least the binding activity andspecificity of the full-length domain.

The term “library” refers to a mixture of heterogeneous polypeptides ornucleic acids. The library is composed of members, each of which has asingle polypeptide or nucleic acid sequence. To this extent, “library”is synonymous with “repertoire.” Sequence differences between librarymembers are responsible for the diversity present in the library. Thelibrary may take the form of a simple mixture of polypeptides or nucleicacids, or may be in the form of organisms or cells, for examplebacteria, viruses, animal or plant cells and the like, transformed witha library of nucleic acids. In one embodiment, each individual organismor cell contains only one or a limited number of library members. In oneembodiment, the nucleic acids are incorporated into expression vectors,in order to allow expression of the polypeptides encoded by the nucleicacids. In an aspect, therefore, a library may take the form of apopulation of host organisms, each organism containing one or morecopies of an expression vector containing a single member of the libraryin nucleic acid form which can be expressed to produce its correspondingpolypeptide member. Thus, the population of host organisms has thepotential to encode a large repertoire of diverse polypeptides.

A “universal framework” is a single antibody framework sequencecorresponding to the regions of an antibody conserved in sequence asdefined by Kabat (“Sequences of Proteins of Immunological Interest”, USDepartment of Health and Human Services) or corresponding to the humangermline immunoglobulin repertoire or structure as defined by Chothiaand Lesk, (1987) J. Mol. Biol. 196:910-917. Libraries and repertoirescan use a single framework, or a set of such frameworks, which has beenfound to permit the derivation of virtually any binding specificitythough variation in the hypervariable regions alone.

As used herein, the term “dose” refers to the quantity of ligandadministered to a subject all at one time (unit dose), or in two or moreadministrations over a defined time interval. For example, dose canrefer to the quantity of ligand (e.g., ligand comprising animmunoglobulin single variable domain that binds target antigen)administered to a subject over the course of one day (24 hours) (dailydose), two days, one week, two weeks, three weeks or one or more months(e.g., by a single administration, or by two or more administrations).The interval between doses can be any desired amount of time.

The phrase, “half-life,” refers to the time taken for the serumconcentration of the ligand (eg, dAb, polypeptide or antagonist) toreduce by 50%, in vivo, for example due to degradation of the ligandand/or clearance or sequestration of the ligand by natural mechanisms.The ligands of the invention are stabilized in vivo and their half-lifeincreased by binding to molecules which resist degradation and/orclearance or sequestration. Typically, such molecules are naturallyoccurring proteins which themselves have a long half-life in vivo. Thehalf-life of a ligand is increased if its functional activity persists,in vivo, for a longer period than a similar ligand which is not specificfor the half-life increasing molecule. For example, a ligand specificfor human serum albumin (HAS) and a target molecule is compared with thesame ligand wherein the specificity to HSA is not present, that is doesnot bind HSA but binds another molecule. For example, it may bind athird target on the cell. Typically, the half-life is increased by 10%,20%, 30%, 40%, 50% or more. Increases in the range of 2×, 3×, 4×, 5×,10×, 20×, 30×, 40×, 50× or more of the half-life are possible.Alternatively, or in addition, increases in the range of up to 30×, 40×,50×, 60×, 70×, 80×, 90×, 100×, 150× of the half-life are possible.

As used herein, “hydrodynamic size” refers to the apparent size of amolecule (e.g., a protein molecule, ligand) based on the diffusion ofthe molecule through an aqueous solution. The diffusion, or motion of aprotein through solution can be processed to derive an apparent size ofthe protein, where the size is given by the “Stokes radius” or“hydrodynamic radius” of the protein particle. The “hydrodynamic size”of a protein depends on both mass and shape (conformation), such thattwo proteins having the same molecular mass may have differinghydrodynamic sizes based on the overall conformation of the protein.

As referred to herein, the term “competes” means that the binding of afirst target to its cognate target binding domain is inhibited in thepresence of a second binding domain that is specific for said cognatetarget. For example, binding may be inhibited sterically, for example byphysical blocking of a binding domain or by alteration of the structureor environment of a binding domain such that its affinity or avidity fora target is reduced. See WO2006038027 for details of how to performcompetition ELISA and competition BiaCore experiments to determinecompetition between first and second binding domains.

Calculations of “homology” or “identity” or “similarity” between twosequences (the terms are used interchangeably herein) are performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Inan embodiment, the length of a reference sequence aligned for comparisonpurposes is at least 30%, or at least 40%, or at least 50%, or at least60%, or at least 70%, 80%, 90%, 100% of the length of the referencesequence. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “homology” is equivalent to amino acidor nucleic acid “identity”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences. Amino acid and nucleotide sequence alignments and homology,similarity or identity, as defined herein may be prepared and determinedusing the algorithm BLAST 2 Sequences, using default parameters(Tatusova, T. A. et al., FEMS Microbiol Lett, 174:187-188 (1999).

Selection Methods

The invention in one embodiment relates to polypeptides and dAbs, e.g.anti-VEGF dAbs, selected by a method of selection of protease resistantpeptides and polypeptides that have a desired biological activity e.g.binding to VEGF. Two selective pressures are used in the method toproduce an efficient process for selecting polypeptides that are highlystable and resistant to protease degradation, and that have desiredbiological activity. As described herein, protease resistant peptidesand polypeptides generally retain biological activity. In contrast,protease sensitive peptides and polypeptides are cleaved or digested byprotease in the methods described herein, therefore, lose theirbiological activity. Accordingly, protease resistant peptides orpolypeptides are generally selected based on their biological activity,such as binding activity.

The methods described herein provide several advantages. For example, asdisclosed and exemplified herein, variable domains, antagonists,peptides or polypeptides that are selected for resistance to proteolyticdegradation by one protease (e.g., trypsin), are also resistant todegradation by other proteases (e.g., elastase, leucozyme). In oneembodiment protease resistance correlates with a higher meltingtemperature (Tm) of the peptide or polypeptide. Higher meltingtemperatures are indicative of more stable variable domains,antagonists, peptides and polypeptides. Resistance to proteasedegradation also correlates in one embodiment with high affinity bindingto target ligands. Thus, the methods described herein provide anefficient way to select, isolate and/or recover variable domains,antagonists, peptides, polypeptides that have a desired biologicalactivity and that are well suited for in vivo therapeutic and/ordiagnostic uses because they are protease resistant and stable. In oneembodiment protease resistance correlates with an improved PK, forexample improved over n variable domain, antagonist, peptide orpolypeptide that is not protease resistant. Improved PK may be animproved AUC (area under the curve) and/or an improved half-life. In oneembodiment protease resistance correlates with an improved stability ofthe variable domain, antagonist, peptide or polypeptide to shear and/orthermal stress and/or a reduced propensity to aggregate duringnebulisation, for example improved over an variable domain, antagonist,peptide or polypeptide that is not protease resistant. In one embodimentprotease resistance correlates with an improved storage stability, forexample improved over an variable domain, antagonist, peptide orpolypeptide that is not protease resistant. In one aspect, one, two,three, four or all of the advantages are provided, the advantages beingresistance to protease degradation, higher Tm and high affinity bindingto target ligand.

Selection Methods

In one aspect, there is provided a method for selecting, isolatingand/or recovering a peptide or polypeptide from a library or arepertoire of peptides and polypeptides (e.g., a display system) that isresistant to degradation by a protease (e.g., one or more proteases). Inone embodiment, the method is a method for selecting, isolating and/orrecovering a polypeptide from a library or a repertoire of peptides andpolypeptides (e.g., a display system) that is resistant to degradationby a protease (e.g., one or more proteases). Generally, the methodcomprises providing a library or repertoire of peptides or polypeptides,combining the library or repertoire with a protease (e.g., trypsin,elastase, leucozyme, pancreatin, sputum) under conditions suitable forprotease activity, and selecting, isolating and/or recovering a peptideor polypeptide that is resistant to degradation by the protease and hasa desired biological activity. Peptides or polypeptides that aredegraded by a protease generally have reduced biological activity orlose their biological activity due to the activity of protease.Accordingly, peptides or polypeptides that are resistant to proteasedegradation can be selected, isolated and/or recovered using the methodbased on their biological activity, such as binding activity (e.g.,binding a general ligand, binding a specific ligand, binding asubstrate), catalytic activity or other biological activity.

The library or repertoire of peptides or polypeptides is combined with aprotease (e.g., one or more proteases) under conditions suitable forproteolytic activity of the protease. Conditions that are suitable forproteolytic activity of protease, and biological preparations ormixtures that contain proteolytic activity, are well-known in the art orcan be readily determined by a person of ordinary skill in the art. Ifdesired, suitable conditions can be identified or optimized, forexample, by assessing protease activity under a range of pH conditions,protease concentrations, temperatures and/or by varying the amount oftime the library or repertoire and the protease are permitted to react.For example, in some embodiments, the ratio (on a mole/mole basis) ofprotease, eg trypsin, to peptide or polypeptide (eg, variable domain) is800 to 80,00 (eg, 8,000 to 80,000) protease:peptide or polypeptide, egwhen 10 micrograms/ml of protease is used, the ratio is 800 to 80,000protease:peptide or polypeptide; or when 100 micrograms/ml of proteaseis used, the ratio is 8,000 to 80,000 protease:peptide or polypeptide.In one embodiment the ratio (on a weight/weight, eg microgram/microgrambasis) of protease (eg, trypsin) to peptide or polypeptide (eg, variabledomain) is 1,600 to 160,000 (eg, 16,000 to 160,000) protease:peptide orpolypeptide eg when 10 micrograms/ml of protease is used, the ratio is1,600 to 160,000 protease:peptide or polypeptide; or when 100micrograms/ml of protease is used, the ratio is 16,000 to 160,000protease:peptide or polypeptide. In one embodiment, the protease is usedat a concentration of at least 100 or 1000 micrograms/ml and theprotease: peptide ratio (on a mole/mole basis) of protease, eg trypsin,to peptide or polypeptide (eg, variable domain) is 8,000 to 80,000protease:peptide or polypeptide. In one embodiment, the protease is usedat a concentration of at least 10 micrograms/ml and the protease:peptide ratio (on a mole/mole basis) of protease, eg trypsin, to peptideor polypeptide (eg, variable domain) is 800 to 80,000 protease:peptideor polypeptide. In one embodiment the ratio (on a weight/weight, egmicrogram/microgram basis) of protease (eg, trypsin) to peptide orpolypeptide (eg, variable domain) is 1600 to 160,000 protease:peptide orpolypeptide eg when C is 10 micrograms/ml; or when C or C′ is 100micrograms/ml, the ratio is 16,000 to 160,000 protease:peptide orpolypeptide. In one embodiment, the concentration (c or c′) is at least100 or 1000 micrograms/ml protease. For testing an individual orisolated peptide or polypeptide (eg, an immunoglobulin variable domain),eg one that has already been isolated from a repertoire or library, aprotease can be added to a solution of peptide or polypeptide in asuitable buffer (e.g., PBS) to produce a peptide or polypeptide/proteasesolution, such as a solution of at least about 0.01% (w/w)protease/peptide or polypeptide, about 0.01% to about 5% (w/w)protease/peptide or polypeptide, about 0.05% to about 5% (w/w)protease/peptide or polypeptide, about 0.1% to about 5% (w/w)protease/peptide or polypeptide, about 0.5% to about 5% (w/w)protease/peptide or polypeptide, about 1% to about 5% (w/w)protease/peptide or polypeptide, at least about 0.01% (w/w)protease/peptide or polypeptide, at least about 0.02% (w/w)protease/peptide or polypeptide, at least about 0.03% (w/w)protease/peptide or polypeptide, at least about 0.04% (w/w)protease/peptide or polypeptide, at least about 0.05% (w/w)protease/peptide or polypeptide, at least about 0.06% (w/w)protease/peptide or polypeptide, at least about 0.07% (w/w)protease/peptide or polypeptide, at least about 0.08% (w/w)protease/peptide or polypeptide, at least about 0.09% (w/w)protease/peptide or polypeptide, at least about 0.1% (w/w)protease/peptide or polypeptide, at least about 0.2% (w/w)protease/peptide polypeptide, at least about 0.3% (w/w) protease/peptideor polypeptide, at least about 0.4% (w/w) protease/peptide orpolypeptide, at least about 0.5% (w/w) protease/peptide or polypeptide,at least about 0.6% (w/w) protease/peptide or polypeptide, at leastabout 0.7% (w/w) protease/peptide or polypeptide, at least about 0.8%(w/w) protease/peptide or polypeptide, at least about 0.9% (w/w)protease/peptide or polypeptide, at least about 1% (w/w)protease/peptide or polypeptide, at least about 2% (w/w)protease/peptide or polypeptide, at least about 3% (w/w)protease/peptide or polypeptide, at least about 4% (w/w)protease/peptide or polypeptide, or about 5% (w/w) protease/peptide orpolypeptide. The mixture can be incubated at a suitable temperature forprotease activity (e.g., room temperature, about 37° C.) and samples canbe taken at time intervals (e.g., at 1 hour, 2 hours, 3 hours, etc.).The samples can be analyzed for protein degradation using any suitablemethod, such as SDS-PAGE analysis or ligand binding, and the results canbe used to establish a time course of degradation.

Any desired protease or proteases can be used in the methods describedherein. For example, a single protease, any desired combination ofdifferent proteases, or any biological preparation, biological extract,or biological homogenate that contains proteolytic activity can be used.It is not necessary that the identity of the protease or proteases thatare used be known. Suitable examples of proteases that can be used aloneor in any desired combination include serine protease, cysteineprotease, aspartate proteases, thiol proteases, matrix metalloprotease,carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B),trypsin, chymotrypsin, pepsin, papain, elastase, leukozyme, pancreatin,thrombin, plasmin, cathepsins (e.g., cathepsin G), proteinase (e.g.,proteinase 1, proteinase 2, proteinase 3), thermolysin, chymosin,enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4, caspase5, caspase 9, caspase 12, caspase 13), calpain, ficain, clostripain,actinidain, bromelain, separase and the like. Suitable biologicalextracts, homogenates and preparations that contains proteolyticactivity include sputum, mucus (e.g., gastric mucus, nasal mucus,bronchial mucus), bronchoalveolar lavage, lung homogenate, lung extract,pancreatic extract, gastric fluid, saliva, tears and the like. Theprotease is used in an amount suitable for proteolytic degradation tooccur. For example, as described herein, protease can be used at about0.01% to about 5% (w/w, protease/peptide or polypeptide). When proteaseis combined with a display system that comprises the repertoire ofpeptides or polypeptides (e.g., a phage display system), for example,the protease can be used at a concentration of about 10 μg/ml to about 3mg/ml, about 10 μg/ml, about 20 μg/ml, about 30 μg/ml, about 40 μg/ml,about 50 μg/ml, about 60 μg/ml, about 70 μg/ml, about 80 μg/ml, about 90μg/ml, about 100 μg/ml, about 200 μg/ml, about 300 μg/ml, about 400μg/ml, about 500 μg/ml, about 600 μg/ml, about 700 μg/ml, about 800μg/ml, about 900 μg/ml, about 1000 μg/ml, about 1.5 mg/ml, about 2mg/ml, about 2.5 mg/ml or about 3 mg/ml.

The protease is incubated with the collection of peptides orpolypeptides (library or repertoire) at a temperature that is suitablefor activity of the protease. For example, the protease and collectionof peptides or polypeptides can be incubated at a temperature of about20° C. to about 40° C. (e.g., at room temperature, about 20° C., about21° C., about 22° C., about 23° C., about 24° C., about 25° C., about26° C., about 27° C., about 28° C., about 29° C., about 30° C., about31° C., about 32° C., about 33° C., about 34° C., about 35° C., about36° C., about 37° C., about 38° C., about 39° C., about 40° C.). Theprotease and the collection of peptides or polypeptides are incubatedtogether for a period of time sufficient for proteolytic degradation tooccur. For example, the collection of peptides or polypeptides can beincubated together with protease for about 30 minutes to about 24 orabout 48 hours. In some examples, the collection of peptides orpolypeptides is incubated together with protease overnight, or for atleast about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours,about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 48hours, or longer.

It is generally desirable, at least in early selection rounds (e.g. whena display system is used), that the protease results in a reduction inthe number of clones that have the desired biological activity that isselected for by at least one order of magnitude, in comparison toselections that do not include incubation with protease. In particularexamples, the amount of protease and conditions used in the methods aresufficient to reduce the number of recovered clones by at least aboutone log (a factor of 10), at least about 2 logs (a factor of 100), atleast about 3 logs (a factor of 1000) or at least about 4 logs (a factorof 10,000). Suitable amounts of protease and incubation conditions thatwill result in the desired reduction in recovered clones can be easilydetermined using conventional methods and/or the guidance providedherein.

The protease and collection of peptides or polypeptides can be combinedand incubated using any suitable method (e.g., in vitro, in vivo or exvivo). For example, the protease and collection of peptides orpolypeptides can be combined in a suitable container and heldstationary, rocked, shaken, swirled or the like, at a temperaturesuitable for protease activity. If desired, the protease and collectionof peptides or polypeptides can be combined in an in vivo or ex vivosystem, such as by introducing the collection of polypeptides (e.g., aphage display library or repertoire) into a suitable animal (e.g., amouse), and after sufficient time for protease activity has passed,recovering the collection of peptides or polypeptides. In anotherexample, an organ or tissue is perfused with the collection ofpolypeptides (e.g., a phage display library or repertoire), and aftersufficient time for protease activity has passed, the collection ofpolypeptides is recovered.

Following incubation, a protease resistant peptide or polypeptide can beselected based on a desired biological activity, such as a bindingactivity. If desired, a protease inhibitor can be added beforeselection. Any suitable protease inhibitor (or combination of two ormore protease inhibitors) that will not substantially interfere with theselection method can be used. Examples of suitable protease inhibitorsinclude, α1-anti-trypsin, α2-macroglobulin, amastatin, antipain,antithrombin III, aprotinin, 4-(2-Aminoethyl)benzenesulfonyl fluoridehydrochloride (AEBSF), (4-Amidino-Phenyl)-Methane-Sulfonyl Fluoride(APMSF), bestatin, benzamidine, chymostatin, 3,4-Dichloroisocoumarin,diisoproply fluorophosphate (DIFP), E-64, ethylenediamine tetraacedicacid (EDTA), elastatinal, leupeptin, N-Ethylmaleimide,phenylmethylsulfonylfluoride (PMSF), pepstatin, 1,10-Phenanthroline,phosphoramidon, serine protease inhibitors,N-tosyl-L-lysine-chloromethyl ketone (TLCK),Na-Tosyl-Phe-chloromethylketone (TPCK) and the like. In addition, manypreparations that contain inhibitors of several classes of proteases arecommercially available (e.g., Roche Complete Protease Inhibitor CocktailTablets™ (Roche Diagnostics Corporation; Indianapolis, Ind., USA), whichinhibits chymotrypsin, thermolysin, papain, pronase, pancreatic extractand trypsin).

A protease resistant peptide or polypeptide can be selected using adesired biological activity selection method, which allows peptides andpolypeptides that have the desired biological activity to bedistinguished from and selected over peptides and polypeptides that donot have the desired biological activity. Generally, peptides orpolypeptides that have been digested or cleaved by protease loose theirbiological activity, while protease resistant peptides or polypeptidesremain functional. Thus, suitable assays for biological activity can beused to select protease resistant peptides or polypeptides. For example,a common binding function (e.g., binding of a general ligand, binding ofa specific ligand, or binding of a substrate) can be assessed using asuitable binding assay (e.g., ELISA, panning) For example, polypeptidesthat bind a target ligand or a generic ligand, such as protein A,protein L or an antibody, can be selected, isolated, and/or recovered bypanning or using a suitable affinity matrix. Panning can be accomplishedby adding a solution of ligand (e.g., generic ligand, target ligand) toa suitable vessel (e.g., tube, petri dish) and allowing the ligand tobecome deposited or coated onto the walls of the vessel. Excess ligandcan be washed away and polypeptides (e.g., a phage display library) canbe added to the vessel and the vessel maintained under conditionssuitable for the polypeptides to bind the immobilized ligand. Unboundpolypeptide can be washed away and bound polypeptides can be recoveredusing any suitable method, such as scraping or lowering the pH, forexample.

When a phage display system is used, binding can be tested in a phageELISA. Phage ELISA may be performed according to any suitable procedure.In one example, populations of phage produced at each round of selectioncan be screened for binding by ELISA to the selected target ligand orgeneric ligand, to identify phage that display protease resistantpeptides or polypeptides. If desired, soluble peptides and polypeptidescan be tested for binding to target ligand or generic ligand, forexample by ELISA using reagents, for example, against a C- or N-terminaltag (see for example Winter et al. (1994) Ann. Rev. Immunology 12,433-55 and references cited therein). The diversity of the selectedphage may also be assessed by gel electrophoresis of PCR products (Markset al. 1991, supra; Nissim et al. 1994 supra), probing (Tomlinson etal., 1992) J. Mol. Biol. 227, 776) or by sequencing of the vector DNA.

In addition to specificity for VEGF, an antagonist or polypeptide (eg, adual specific ligand) comprising an anti-VEGF protease resistantpolypeptide (e.g., single antibody variable domain) can have bindingspecificity for a generic ligand or any desired target ligand, such ashuman or animal proteins, including cytokines, growth factors, cytokinereceptors, growth factor receptors, enzymes (e.g., proteases),co-factors for enzymes, DNA binding proteins, lipids and carbohydrates.

In some embodiments, the protease resistant peptide or polypeptide (eg,dAb) or antagonist binds VEGF in pulmonary tissue. In one embodiment,the antagonist or polypeptide also binds a further target in pulmonarytissue.

When a display system (e.g., a display system that links coding functionof a nucleic acid and functional characteristics of the peptide orpolypeptide encoded by the nucleic acid) is used in the methodsdescribed herein it may be frequently advantageous to amplify orincrease the copy number of the nucleic acids that encode the selectedpeptides or polypeptides. This provides an efficient way of obtainingsufficient quantities of nucleic acids and/or peptides or polypeptidesfor additional rounds of selection, using the methods described hereinor other suitable methods, or for preparing additional repertoires(e.g., affinity maturation repertoires). Thus, in some embodiments, themethods comprise using a display system (e.g., that links codingfunction of a nucleic acid and functional characteristics of the peptideor polypeptide encoded by the nucleic acid, such as phage display) andfurther comprises amplifying or increasing the copy number of a nucleicacid that encodes a selected peptide or polypeptide. Nucleic acids canbe amplified using any suitable methods, such as by phage amplification,cell growth or polymerase chain reaction.

The methods described herein can be used as part of a program to isolateprotease resistant peptides or polypeptides, eg dAbs, that can comprise,if desired, other suitable selection methods. In these situations, themethods described herein can be employed at any desired point in theprogram, such as before or after other selection methods are used. Themethods described herein can also be used to provide two or more roundsof selection, as described and exemplified herein.

In one example, there is provided a method for selecting a peptide orpolypeptide (eg, a dAb) that specifically binds VEGF and is resistant todegradation by trypsin, comprising providing a library or repertoire ofthe peptides or polypeptides, combining the library or repertoire withtrypsin under conditions suitable for proteolytic digestion by trypsin,and selecting, isolating and/or recovering a peptide or polypeptide thatis resistant to degradation by trypsin and specifically binds VEGF.

In particular embodiments, there is provided a method for selecting animmunoglobulin single variable domain (a dAb) that is resistant todegradation by trypsin and specifically binds VEGF. In theseembodiments, a library or repertoire comprising dAbs is provided andcombined with trypsin (or a biological preparation, extract orhomogenate comprising trypsin) under conditions suitable for proteolyticdigestion by trypsin. Trypsin resistant dAbs are selected that bindVEGF. For example, the trypsin resistant dAb is not substantiallydegraded when incubated at 37° C. in a 0.04% (w/w) solution of trypsinfor a period of at least about 2 hours. In one embodiment, the trypsinresistant dAb is not substantially degraded when incubated at 37° C. ina 0.04% (w/w) solution of trypsin for a period of at least about 3hours. In one embodiment, the trypsin resistant dAb is not substantiallydegraded when incubated at 37° C. in a 0.04% (w/w) solution of trypsinfor a period of at least about 4 hours, at least about 5 hours, at leastabout 6 hours, at least about 7 hours, at least about 8 hours, at leastabout 9 hours, at least about 10 hours, at least about 11 hours, or atleast about 12 hours.

In an exemplary embodiment, there is provided a method for selecting animmunoglobulin single variable domain (a dAb) that is resistant todegradation by trypsin and specifically binds VEGF. The method comprisesproviding a phage display system comprising a repertoire of polypeptidesthat comprise an immunoglobulin single variable domain, combining thephage display system with trypsin (100 μg/ml) and incubating the mixtureat about 37° C., for example overnight (e.g., about 12-16 hours), andthen selecting phage that display a dAb that specifically bind VEGF.

In another example, the method is for selecting a peptide orpolypeptide, eg a dAb, that is resistant to degradation by elastase,comprising providing a library or repertoire of peptides orpolypeptides, combining the library or repertoire with elastase (or abiological preparation, extract or homogenate comprising elastase) underconditions suitable for proteolytic digestion by elastase, andselecting, isolating and/or recovering a peptide or polypeptide that isresistant to degradation by elastase and has VEGF binding activity.

In particular embodiments, there is provided a method for selecting animmunoglobulin single variable domain (a dAb) that is resistant todegradation by elastase and binds VEGF. In these embodiments, a libraryor repertoire comprising dAbs is provided and combined with elastase (ora biological preparation, extract or homogenate comprising elastase)under conditions suitable for proteolytic digestion by elastase.Elastase resistant dAbs are selected that specifically bind VEGF. Forexample, the elastase resistant dAb is not substantially degraded whenincubated at 37° C. in a 0.04% (w/w) solution of elastase for a periodof at least about 2 hours. In one embodiment, the elastase resistant dAbis not substantially degraded when incubated at 37° C. in a 0.04% (w/w)solution of elastase for a period of at least about 12 hours. In oneembodiment, the elastase resistant dAb is not substantially degradedwhen incubated at 37° C. in a 0.04% (w/w) solution of elastase for aperiod of at least about 24 hours, at least about 36 hours, or at leastabout 48 hours.

In an embodiment, there is provided a method for selecting animmunoglobulin single variable domain (a dAb) that is resistant todegradation by elastase and binds VEGF. The method comprises providing aphage display system comprising a repertoire of polypeptides thatcomprise an immunoglobulin single variable domain, combining the phagedisplay system with elastase (about 100 μg/ml) and incubating themixture at about 37° C., for example, overnight (e.g., about 12-16hours), and then selecting phage that display a dAb that specificallybind VEGF.

In one example, there is provided a method for selecting a peptide orpolypeptide (eg, a dAb) that is resistant to degradation by leucozyme,comprising providing a library or repertoire of peptides orpolypeptides, combining the library or repertoire with leucozyme (or abiological preparation, extract or homogenate comprising leucozyme)under conditions suitable for proteolytic digestion by leucozyme, andselecting, isolating and/or recovering a peptide or polypeptide that isresistant to degradation by leucozyme and has specific VEGF bindingactivity.

In particular embodiments, there is provided a method for selecting animmunoglobulin single variable domain (a dAb) that is resistant todegradation by leucozyme and binds VEGF. In these embodiments, a libraryor repertoire comprising dAbs is provided and combined with leucozyme(or a biological preparation, extract or homogenate comprisingleucozyme) under conditions suitable for proteolytic digestion byleucozyme. Leucozyme resistant dAbs are selected that specifically bindVEGF. For example, the leucozyme resistant dAb is not substantiallydegraded when incubated at 37° C. in a 0.04% (w/w) solution of leucozymefor a period of at least about 2 hours. In one embodiment, the leucozymeresistant dAb is not substantially degraded when incubated at 37° C. ina 0.04% (w/w) solution of leucozyme for a period of at least about 12hours. In one embodiment, the leucozyme resistant dAb is notsubstantially degraded when incubated at 37° C. in a 0.04% (w/w)solution of leucozyme for a period of at least about 24 hours, at leastabout 36 hours, or at least about 48 hours.

In an embodiment, there is provided a method for selecting animmunoglobulin single variable domain (a dAb) that is resistant todegradation by leucozyme and specifically binds VEGF. The methodcomprises providing a phage display system comprising a repertoire ofpolypeptides that comprise an immunoglobulin single variable domain,combining the phage display system with leucozyme (about 100 μg/ml) andincubating the mixture at about 37° C., for example, overnight (e.g.,about 12-16 hours), and then selecting phage that display a dAb thatspecifically bind VEGF.

In another aspect, there is provided a method of producing a repertoireof protease resistant peptides or polypeptides (eg, dAbs). The methodcomprises providing a repertoire of peptides or polypeptides; combiningthe repertoire of peptides or polypeptides and a protease under suitableconditions for protease activity; and recovering a plurality of peptidesor polypeptides that specifically bind VEGF, whereby a repertoire ofprotease resistant peptides or polypeptides is produced. Proteases,display systems, conditions for protease activity, and methods forselecting peptides or polypeptides that are suitable for use in themethod are described herein with respect to the other methods.

In some embodiments, a display system (e.g., a display system that linkscoding function of a nucleic acid and functional characteristics of thepeptide or polypeptide encoded by the nucleic acid) that comprises arepertoire of peptides or polypeptides is used, and the method furthercomprises amplifying or increasing the copy number of the nucleic acidsthat encode the plurality of selected peptides or polypeptides. Nucleicacids can be amplified using any suitable method, such as by phageamplification, cell growth or polymerase chain reaction.

In particular embodiment, there is provided a method of producing arepertoire of protease resistant polypeptides that comprise anti-VEGFdAbs. The method comprises providing a repertoire of polypeptides thatcomprise anti-VEGF dAbs; combining the repertoire of peptides orpolypeptides and a protease (e.g., trypsin, elastase, leucozyme) undersuitable conditions for protease activity; and recovering a plurality ofpolypeptides that comprise dAbs that have binding specificity for VEGF.The method can be used to produce a naïve repertoire, or a repertoirethat is biased toward a desired binding specificity, such as an affinitymaturation repertoire based on a parental dAb that has bindingspecificity for VEGF.

Polypeptide Display Systems

In one embodiment, the repertoire or library of peptides or polypeptidesprovided for use in the methods described herein comprise a suitabledisplay system. The display system may resist degradation by protease(e.g., a single protease or a combination of proteases, and anybiological extract, homogenate or preparation that contains proteolyticactivity (e.g., sputum, mucus (e.g., gastric mucus, nasal mucus,bronchial mucus), bronchoalveolar lavage, lung homogenate, lung extract,pancreatic extract, gastric fluid, saliva, tears and the like). Thedisplay system and the link between the display system and the displayedpolypeptide is in one embodiment at least as resistant to protease asthe most stable peptides or polypeptides of the repertoire. This allowsa nucleic acid that encodes a selected displayed polypeptide to beeasily isolated and/or amplified.

In one example, a protease resistant peptide or polypeptide, eg a dAb,can be selected, isolated and/or recovered from a repertoire of peptidesor polypeptides that is in solution, or is covalently or noncovalentlyattached to a suitable surface, such as plastic or glass (e.g.,microtiter plate, polypeptide array such as a microarray). For examplean array of peptides on a surface in a manner that places each distinctlibrary member (e.g., unique peptide sequence) at a discrete, predefinedlocation in the array can be used. The identity of each library memberin such an array can be determined by its spatial location in the array.The locations in the array where binding interactions between a targetligand, for example, and reactive library members occur can bedetermined, thereby identifying the sequences of the reactive members onthe basis of spatial location. (See, e.g., U.S. Pat. No. 5,143,854, WO90/15070 and WO 92/10092.)

In one embodiment, the methods employ a display system that links thecoding function of a nucleic acid and physical, chemical and/orfunctional characteristics of the polypeptide encoded by the nucleicacid. Such a display system can comprise a plurality of replicablegenetic packages, such as bacteriophage or cells (bacteria). In oneembodiment, the display system comprises a library, such as abacteriophage display library.

A number of suitable bacteriophage display systems (e.g., monovalentdisplay and multivalent display systems) have been described. (See,e.g., Griffiths et al., U.S. Pat. No. 6,555,313 B1 (incorporated hereinby reference); Johnson et al., U.S. Pat. No. 5,733,743 (incorporatedherein by reference); McCafferty et al., U.S. Pat. No. 5,969,108(incorporated herein by reference); Mulligan-Kehoe, U.S. Pat. No.5,702,892 (Incorporated herein by reference); Winter, G. et al., Annu.Rev. Immunol. 12:433-455 (1994); Soumillion, P. et al., Appl. Biochem.Biotechnol. 47(2-3):175-189 (1994); Castagnoli, L. et al., Comb. Chem.High Throughput Screen, 4(2):121-133 (2001).) The peptides orpolypeptides displayed in a bacteriophage display system can bedisplayed on any suitable bacteriophage, such as a filamentous phage(e.g., fd, M13, F1), a lytic phage (e.g., T4, T7, lambda), or an RNAphage (e.g., MS2), for example.

Generally, a library of phage that displays a repertoire of peptides orphage polypeptides, as fusion proteins with a suitable phage coatprotein (e.g., fd pIII protein), is produced or provided. The fusionprotein can display the peptides or polypeptides at the tip of the phagecoat protein, or if desired at an internal position. For example, thedisplayed peptide or polypeptide can be present at a position that isamino-terminal to domain 1 of pIII. (Domain 1 of pIII is also referredto as N1.) The displayed polypeptide can be directly fused to pIII(e.g., the N-terminus of domain 1 of pIII) or fused to pIII using alinker. If desired, the fusion can further comprise a tag (e.g., mycepitope, His tag). Libraries that comprise a repertoire of peptides orpolypeptides that are displayed as fusion proteins with a phage coatprotein can be produced using any suitable methods, such as byintroducing a library of phage vectors or phagemid vectors encoding thedisplayed peptides or polypeptides into suitable host bacteria, andculturing the resulting bacteria to produce phage (e.g., using asuitable helper phage or complementing plasmid if desired). The libraryof phage can be recovered from the culture using any suitable method,such as precipitation and centrifugation.

The display system can comprise a repertoire of peptides or polypeptidesthat contains any desired amount of diversity. For example, therepertoire can contain peptides or polypeptides that have amino acidsequences that correspond to naturally occurring polypeptides expressedby an organism, group of organisms (eg, a repertoire of sequences ofV_(HH) dAbs isolated from a Camelid), desired tissue or desired celltype, or can contain peptides or polypeptides that have random orrandomized amino acid sequences. If desired, the polypeptides can sharea common core or scaffold. The polypeptides in such a repertoire orlibrary can comprise defined regions of random or randomized amino acidsequence and regions of common amino acid sequence. In certainembodiments, all or substantially all polypeptides in a repertoire areof a desired type, such as a desired enzyme (e.g., a polymerase) or adesired antigen-binding fragment of an antibody (e.g., human V_(H) orhuman V_(L)). In embodiments, the polypeptide display system comprises arepertoire of polypeptides wherein each polypeptide comprises anantibody variable domain. For example, each polypeptide in therepertoire can contain a V_(H), a V_(L) or an Fv (e.g., a single chainFv).

Amino acid sequence diversity can be introduced into any desired regionof a peptide or polypeptide or scaffold using any suitable method. Forexample, amino acid sequence diversity can be introduced into a targetregion, such as a complementarity determining region of an antibodyvariable domain or a hydrophobic domain, by preparing a library ofnucleic acids that encode the diversified polypeptides using anysuitable mutagenesis methods (e.g., low fidelity PCR,oligonucleotide-mediated or site directed mutagenesis, diversificationusing NNK codons) or any other suitable method. If desired, a region ofa polypeptide to be diversified can be randomized.

The size of the polypeptides that make up the repertoire is largely amatter of choice and uniform polypeptide size is not required. In oneembodiment, the polypeptides in the repertoire have at least tertiarystructure (form at least one domain).

Selection/Isolation/Recovery

A protease resistant peptide or polypeptide (e.g., a population ofprotease resistant polypeptides) can be selected, isolated and/orrecovered from a repertoire or library (e.g., in a display system) usingany suitable method. In one embodiment, a protease resistant polypeptideis selected or isolated based on a selectable characteristic (e.g.,physical characteristic, chemical characteristic, functionalcharacteristic). Suitable selectable functional characteristics includebiological activities of the peptides or polypeptides in the repertoire,for example, binding to a generic ligand (e.g., a superantigen), bindingto a target ligand (e.g., an antigen, an epitope, a substrate), bindingto an antibody (e.g., through an epitope expressed on a peptide orpolypeptide), and catalytic activity. (See, e.g., Tomlinson et al., WO99/20749; WO 01/57065; WO 99/58655). In one embodiment, the selection isbased on specific binding to VEGF. In another embodiment, selection ison the basis of the selected functional characteristic to produce asecond repertoire in which members are protease resistant, followed byselection of a member from the second repertoire that specifically bindsVEGF.

In some embodiments, the protease resistant peptide or polypeptide isselected and/or isolated from a library or repertoire of peptides orpolypeptides in which substantially all protease resistant peptides orpolypeptides share a common selectable feature. For example, theprotease resistant peptide or polypeptide can be selected from a libraryor repertoire in which substantially all protease resistant peptides orpolypeptides bind a common generic ligand, bind a common target ligand,bind (or are bound by) a common antibody, or possess a common catalyticactivity. This type of selection is particularly useful for preparing arepertoire of protease resistant peptides or polypeptides that are basedon a parental peptide or polypeptide that has a desired biologicalactivity, for example, when performing affinity maturation of animmunoglobulin single variable domain.

Selection based on binding to a common generic ligand can yield acollection or population of peptides or polypeptides that contain all orsubstantially all of the protease resistant peptides or polypeptidesthat were components of the original library or repertoire. For example,peptides or polypeptides that bind a target ligand or a generic ligand,such as protein A, protein L or an antibody, can be selected, isolatedand/or recovered by panning or using a suitable affinity matrix. Panningcan be accomplished by adding a solution of ligand (e.g., genericligand, target ligand) to a suitable vessel (e.g., tube, petri dish) andallowing the ligand to become deposited or coated onto the walls of thevessel. Excess ligand can be washed away and peptides or polypeptides(e.g., a repertoire that has been incubated with protease) can be addedto the vessel and the vessel maintained under conditions suitable forpeptides or polypeptides to bind the immobilized ligand. Unboundpeptides or polypeptides can be washed away and bound peptides orpolypeptides can be recovered using any suitable method, such asscraping or lowering the pH, for example.

Suitable ligand affinity matrices generally contain a solid support orbead (e.g., agarose) to which a ligand is covalently or noncovalentlyattached. The affinity matrix can be combined with peptides orpolypeptides (e.g., a repertoire that has been incubated with protease)using a batch process, a column process or any other suitable processunder conditions suitable for binding of peptides or polypeptides to theligand on the matrix. Peptides or polypeptides that do not bind theaffinity matrix can be washed away and bound peptides or polypeptidescan be eluted and recovered using any suitable method, such as elutionwith a lower pH buffer, with a mild denaturing agent (e.g., urea), orwith a peptide that competes for binding to the ligand. In one example,a biotinylated target ligand is combined with a repertoire underconditions suitable for peptides or polypeptides in the repertoire tobind the target ligand (VEGF). Bound peptides or polypeptides arerecovered using immobilized avidin or streptavidin (e.g., on a bead).

In some embodiments, the generic ligand is an antibody or antigenbinding fragment thereof. Antibodies or antigen binding fragments thatbind structural features of peptides or polypeptides that aresubstantially conserved in the peptides or polypeptides of a library orrepertoire are particularly useful as generic ligands. Antibodies andantigen binding fragments suitable for use as ligands for isolating,selecting and/or recovering protease resistant peptides or polypeptidescan be monoclonal or polyclonal and can be prepared using any suitablemethod.

Libraries/Repertoires

In other aspects, there are provided repertoires of protease resistantpeptides and polypeptides, to libraries that encode protease resistantpeptides and polypeptides, and to methods for producing such librariesand repertoires.

Libraries that encode and/or contain protease resistant peptides andpolypeptides can be prepared or obtained using any suitable method. Thelibrary can be designed to encode protease resistant peptides orpolypeptides based on a peptide or polypeptide of interest (e.g., ananti-VEGF peptide or polypeptide selected from a library) or can beselected from another library using the methods described herein. Forexample, a library enriched in protease resistant polypeptides can beprepared using a suitable polypeptide display system.

In one example, a phage display library comprising a repertoire ofdisplayed polypeptides comprising immunoglobulin single variable domains(e.g., V_(H), Vk, Vλ) is combined with a protease under conditionssuitable for protease activity, as described herein. Protease resistantpolypeptides are recovered based on a desired biological activity, suchas a binding activity (e.g., binding generic ligand, binding targetligand) thereby yielding a phage display library enriched in proteaseresistant polypeptides. In one embodiment, the recovery is on the basisof binding generic ligand to yield an enriched library, followed byselection of an anti-VEGF member of that library based on specificbinding to VEGF.

In another example, a phage display library comprising a repertoire ofdisplayed polypeptides comprising immunoglobulin single variable domains(e.g., V_(H), Vκ, Vλ) is first screened to identify members of therepertoire that have binding specificity for a desired target antigen(e.g. VEGF). A collection of polypeptides having the desired bindingspecificity are recovered and the collection is combined with proteaseunder conditions suitable for proteolytic activity, as described herein.A collection of protease resistant polypeptides that have the desiredtarget binding specificity is recovered, yielding a library enriched inprotease resistant and high affinity polypeptides. As described hereinin an embodiment, protease resistance in this selection methodcorrelates with high affinity binding.

Libraries that encode a repertoire of a desired type of polypeptides canreadily be produced using any suitable method. For example, a nucleicacid sequence that encodes a desired type of polypeptide (e.g., apolymerase, an immunoglobulin variable domain) can be obtained and acollection of nucleic acids that each contain one or more mutations canbe prepared, for example by amplifying the nucleic acid using anerror-prone polymerase chain reaction (PCR) system, by chemicalmutagenesis (Deng et al., J. Biol. Chem., 269:9533 (1994)) or usingbacterial mutator strains (Low et al., J. Mol. Biol., 260:359 (1996)).

In other embodiments, particular regions of the nucleic acid can betargeted for diversification. Methods for mutating selected positionsare also well known in the art and include, for example, the use ofmismatched oligonucleotides or degenerate oligonucleotides, with orwithout the use of PCR. For example, synthetic antibody libraries havebeen created by targeting mutations to the antigen binding loops. Randomor semi-random antibody H3 and L3 regions have been appended to germlineimmunoblulin V gene segments to produce large libraries with unmutatedframework regions (Hoogenboom and Winter (1992) supra; Nissim et al.(1994) supra; Griffiths et al. (1994) supra; DeKruif et al. (1995)supra). Such diversification has been extended to include some or all ofthe other antigen binding loops (Crameri et al. (1996) Nature Med.,2:100; Riechmann et al. (1995) Bio/Technology, 13:475; Morphosys, WO97/08320, supra). In other embodiments, particular regions of thenucleic acid can be targeted for diversification by, for example, atwo-step PCR strategy employing the product of the first PCR as a“mega-primer.” (See, e.g., Landt, O. et al., Gene 96:125-128 (1990).)Targeted diversification can also be accomplished, for example, by SOEPCR. (See, e.g., Horton, R. M. et al., Gene 77:61-68 (1989).)

Sequence diversity at selected positions can be achieved by altering thecoding sequence which specifies the sequence of the polypeptide suchthat a number of possible amino acids (e.g., all 20 or a subset thereof)can be incorporated at that position. Using the IUPAC nomenclature, themost versatile codon is NNK, which encodes all amino acids as well asthe TAG stop codon. The NNK codon may be used in order to introduce therequired diversity. Other codons which achieve the same ends are also ofuse, including the NNN codon, which leads to the production of theadditional stop codons TGA and TAA. Such a targeted approach can allowthe full sequence space in a target area to be explored.

The libraries can comprise protease resistant antibody polypeptides thathave a known main-chain conformation. (See, e.g., Tomlinson et al., WO99/20749.)

Libraries can be prepared in a suitable plasmid or vector. As usedherein, vector refers to a discrete element that is used to introduceheterologous DNA into cells for the expression and/or replicationthereof. Any suitable vector can be used, including plasmids (e.g.,bacterial plasmids), viral or bacteriophage vectors, artificialchromosomes and episomal vectors. Such vectors may be used for simplecloning and mutagenesis, or an expression vector can be used to driveexpression of the library. Vectors and plasmids usually contain one ormore cloning sites (e.g., a polylinker), an origin of replication and atleast one selectable marker gene. Expression vectors can further containelements to drive transcription and translation of a polypeptide, suchas an enhancer element, promoter, transcription termination signal,signal sequences, and the like. These elements can be arranged in such away as to be operably linked to a cloned insert encoding a polypeptide,such that the polypeptide is expressed and produced when such anexpression vector is maintained under conditions suitable for expression(e.g., in a suitable host cell).

Cloning and expression vectors generally contain nucleic acid sequencesthat enable the vector to replicate in one or more selected host cells.Typically in cloning vectors, this sequence is one that enables thevector to replicate independently of the host chromosomal DNA andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (e.g. SV40, adenovirus) are usefulfor cloning vectors in mammalian cells. Generally, the origin ofreplication is not needed for mammalian expression vectors, unless theseare used in mammalian cells able to replicate high levels of DNA, suchas COS cells.

Cloning or expression vectors can contain a selection gene also referredto as selectable marker. Such marker genes encode a protein necessaryfor the survival or growth of transformed host cells grown in aselective culture medium. Host cells not transformed with the vectorcontaining the selection gene will therefore not survive in the culturemedium. Typical selection genes encode proteins that confer resistanceto antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexateor tetracycline, complement auxotrophic deficiencies, or supply criticalnutrients not available in the growth media.

Suitable expression vectors can contain a number of components, forexample, an origin of replication, a selectable marker gene, one or moreexpression control elements, such as a transcription control element(e.g., promoter, enhancer, terminator) and/or one or more translationsignals, a signal sequence or leader sequence, and the like. Expressioncontrol elements and a signal or leader sequence, if present, can beprovided by the vector or other source. For example, the transcriptionaland/or translational control sequences of a cloned nucleic acid encodingan antibody chain can be used to direct expression.

A promoter can be provided for expression in a desired host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding an antibody, antibodychain or portion thereof, such that it directs transcription of thenucleic acid. A variety of suitable promoters for procaryotic (e.g., theβ-lactamase and lactose promoter systems, alkaline phosphatase, thetryptophan (trp) promoter system, lac, tac, T3, T7 promoters for E.coli) and eucaryotic (e.g., simian virus 40 early or late promoter, Roussarcoma virus long terminal repeat promoter, cytomegalovirus promoter,adenovirus late promoter, EG-1a promoter) hosts are available.

In addition, expression vectors typically comprise a selectable markerfor selection of host cells carrying the vector, and, in the case of areplicable expression vector, an origin of replication. Genes encodingproducts which confer antibiotic or drug resistance are commonselectable markers and may be used in procaryotic (e.g., β-lactamasegene (ampicillin resistance), Tet gene for tetracycline resistance) andeucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated.

Suitable expression vectors for expression in prokaryotic (e.g.,bacterial cells such as E. coli) or mammalian cells include, forexample, a pET vector (e.g., pET-12a, pET-36, pET-37, pET-39, pET-40,Novagen and others), a phage vector (e.g., pCANTAB 5 E, Pharmacia),pRIT2T (Protein A fusion vector, Pharmacia), pCDM8, pcDNA1.1/amp,pcDNA3.1, pRc/RSV, pEF-1 (Invitrogen, Carlsbad, Calif.), pCMV-SCRIPT,pFB, pSG5, pXT1 (Stratagene, La Jolla, Calif.), pCDEF3 (Goldman, L. A.,et al., Biotechniques, 21:1013-1015 (1996)), pSVSPORT (GibcoBRL,Rockville, Md.), pEF-Bos (Mizushima, S., et al., Nucleic Acids Res.,18:5322 (1990)) and the like. Expression vectors which are suitable foruse in various expression hosts, such as prokaryotic cells (E. coli),insect cells (Drosophila Schnieder S2 cells, Sf9), yeast (P.methanolica, P. pastoris, S. cerevisiae) and mammalian cells (eg, COScells) are available.

Examples of vectors are expression vectors that enable the expression ofa nucleotide sequence corresponding to a polypeptide library member.Thus, selection with generic and/or target ligands can be performed byseparate propagation and expression of a single clone expressing thepolypeptide library member. As described above, the selection displaysystem may be bacteriophage display. Thus, phage or phagemid vectors maybe used. Example vectors are phagemid vectors which have an E. coli.origin of replication (for double stranded replication) and also a phageorigin of replication (for production of single-stranded DNA). Themanipulation and expression of such vectors is well known in the art(Hoogenboom and Winter (1992) supra; Nissim et al. (1994) supra).Briefly, the vector can contain a β-lactamase gene to confer selectivityon the phagemid and a lac promoter upstream of an expression cassettethat can contain a suitable leader sequence, a multiple cloning site,one or more peptide tags, one or more TAG stop codons and the phageprotein pIII. Thus, using various suppressor and non-suppressor strainsof E. coli and with the addition of glucose, iso-propylthio-β-D-galactoside (IPTG) or a helper phage, such as VCS M13, thevector is able to replicate as a plasmid with no expression, producelarge quantities of the polypeptide library member only or productphage, some of which contain at least one copy of the polypeptide-pIIIfusion on their surface.

The libraries and repertoires described herein can contain antibodyformats. For example, the polypeptide contained within the libraries andrepertoires can be whole separate V_(H) or V_(L) domains, any of whichare either modified or unmodified. scFv fragments, as well as otherantibody polypeptides, can be readily produced using any suitablemethod. A number of suitable antibody engineering methods are well knownin the art. For example, a scFv can be formed by linking nucleic acidsencoding two variable domains with a suitable oligonucleotide thatencodes an appropriate linker peptide, such as (Gly-Gly-Gly-Gly-Ser)₃ orother suitable linker peptides. The linker bridges the C-terminal end ofthe first V region and the N-terminal end of the second V region.Similar techniques for the construction of other antibody formats, suchas Fv, Fab and F(ab′)₂ fragments can be used. To format Fab and F(ab′)₂fragments, V_(H) and V_(L) polypeptides can be combined with constantregion segments, which may be isolated from rearranged genes, germline Cgenes or synthesized from antibody sequence data. A library orrepertoire described herein can be a V_(H) or V_(L) library orrepertoire.

The polypeptides comprising a protease resistant variable domain maycomprise a target ligand (e.g. VEGF) binding site and a generic ligandbinding site. In certain embodiments, the generic ligand binding site isa binding site for a superantigen, such as protein A, protein L orprotein G. The variable domains can be based on any desired variabledomain, for example a human VH (e.g., V_(H) 1a, V_(H) 1b, V_(H) 2, V_(H)3, V_(H) 4, V_(H) 5, V_(H) 6), a human Vλ (e.g., VλI, VλII, VλIII, VλIV,VλV, VλVI or Vκ1) or a human VK (e.g., Vη2, Vκ3, Vκ4, Vκ5, Vκ6, Vκ7,Vκ8, Vκ9 or Vκ10) or a Camelid V_(HH), optionally that has beenhumanized.

Nucleic Acids, Host Cells and Methods for Producing Protease ResistantPolypeptides

The invention relates to isolated and/or recombinant nucleic acidsencoding protease resistant peptides or polypeptides e.g., that areselectable or selected by the methods described herein.

Nucleic acids referred to herein as “isolated” are nucleic acids whichhave been separated away from other material (e.g., other nucleic acidssuch as genomic DNA, cDNA and/or RNA) in its original environment (e.g.,in cells or in a mixture of nucleic acids such as a library). Anisolated nucleic acid can be isolated as part of a vector (e.g., aplasmid).

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, includingmethods which rely upon artificial recombination, such as cloning into avector or chromosome using, for example, restriction enzymes, homologousrecombination, viruses and the like, and nucleic acids prepared usingthe polymerase chain reaction (PCR).

The invention also relates to a recombinant host cell which comprises a(one or more) recombinant nucleic acid or expression constructcomprising a nucleic acid encoding a protease resistant peptide orpolypeptide, e.g., a peptide or polypeptide selectable or selected bythe methods described herein. There is also provided a method ofpreparing a protease resistant peptide or polypeptide, comprisingmaintaining a recombinant host cell of the invention under conditionsappropriate for expression of a protease resistant peptide orpolypeptide. The method can further comprise the step of isolating orrecovering the protease resistant peptide or polypeptide, if desired.

For example, a nucleic acid molecule (i.e., one or more nucleic acidmolecules) encoding a protease resistant peptide or polypeptide, or anexpression construct (i.e., one or more constructs) comprising suchnucleic acid molecule(s), can be introduced into a suitable host cell tocreate a recombinant host cell using any method appropriate to the hostcell selected (e.g., transformation, transfection, electroporation,infection), such that the nucleic acid molecule(s) are operably linkedto one or more expression control elements (e.g., in a vector, in aconstruct created by processes in the cell, integrated into the hostcell genome). The resulting recombinant host cell can be maintainedunder conditions suitable for expression (e.g., in the presence of aninducer, in a suitable animal, in suitable culture media supplementedwith appropriate salts, growth factors, antibiotics, nutritionalsupplements, etc.), whereby the encoded peptide or polypeptide isproduced. If desired, the encoded peptide or polypeptide can be isolatedor recovered (e.g., from the animal, the host cell, medium, milk). Thisprocess encompasses expression in a host cell of a transgenic animal(see, e.g., WO 92/03918, GenPharm International).

The protease resistant peptide or polypeptide selected by the methoddescribed herein can also be produced in a suitable in vitro expressionsystem, by chemical synthesis or by any other suitable method.

Polypeptides, dAbs & Antagonists

As described and exemplified herein, protease resistant dAbs of theinvention generally bind their target ligand with high affinity. Thus,in another aspect, there is provided a method for selecting, isolatingand/or recovering a polypeptide or dAb of the invention that binds VEGFwith high affinity. Generally, the method comprises providing a libraryor repertoire of peptides or polypeptides (eg dAbs), combining thelibrary or repertoire with a protease (e.g., trypsin, elastase,leucozyme, pancreatin, sputum) under conditions suitable for proteaseactivity, and selecting, isolating and/or recovering a peptide orpolypeptide that binds a ligand (e.g., target ligand). Because thelibrary or repertoire has been exposed to protease under conditionswhere protease sensitive peptides or polypeptides will be digested, theactivity of protease can eliminate the less stable polypeptides thathave low binding affinity, and thereby produce a collection of highaffinity binding peptides or polypeptides.

For example, the polypeptide or dAb of the invention can bind VEGF withan affinity (KD; KD=K_(off) (kd)/K_(on) (ka) as determined by surfaceplasmon resonance) of 300 nM to 1 pM (i.e., 3×10⁻⁷ to 5×10⁻¹²M), e.g. 50nM to 1 pM, e.g. 5 nM to 1 pM and e.g. 1 nM to 1 pM; for example K_(D)of 1×10⁻⁷ M or less, e.g. 1×10⁻⁸ M or less, e.g. 1×10⁻⁹ M or less, e.g.1×10⁻¹⁰ M or less and e.g. 1×10⁻¹¹ M or less; and/or a K_(off) rateconstant of 5×10⁻¹ s⁻¹ to 1×10⁻⁷s⁻¹, e.g. 1×10⁻² s⁻¹ to 1×10⁻⁶ s⁻¹, e.g.5×10⁻³ s⁻¹ to 1×10⁻⁵ s⁻¹, for example 5×10⁻¹ s⁻¹ or less, e.g. 1×10⁻²s⁻¹ or less, e.g. 1×10⁻³ s¹ or less, e.g. 1×10⁻⁴ s⁻¹ or less, e.g.1×10⁻⁵ s¹ or less, and e.g. 1×10⁻⁶s⁻¹ or less as determined by surfaceplasmon resonance.

Although we are not bound by any particular theory, peptides andpolypeptides that are resistant to proteases are believed to have alower entropy and/or a higher stabilization energy. Thus, thecorrelation between protease resistance and high affinity binding may berelated to the compactness and stability of the surfaces of the peptidesand polypeptides and dAbs selected by the method described herein.

In one embodiment, the polypeptide, dAb or antagonist of the inventioninhibits binding of VEGF at a concentration 50 (IC50) of IC50 of about 1μM or less, about 500 nM or less, about 100 nM or less, about 75 nM orless, about 50 nM or less, about 10 nM or less or about 1 nM or less.

In certain embodiments, the polypeptide, dAb or antagonist specificallybinds VEGF, eg, human VEGF, and dissociates from human VEGF with adissociation constant (K_(D)) of 300 nM to 1 μM or 300 nM to 5 pM or 50nM to 1 pM or 50 nM to 5 pM or 50 nM to 20 pM or about 10 pM or about 15pM or about 20 pM as determined by surface plasmon resonance. In certainembodiments, the polypeptide, dAb or antagonist specifically binds VEGF,eg, human VEGF, and dissociates from human VEGF with a K_(off) rateconstant of 5×10⁻¹ s⁻¹ to 1×10⁻⁷ s⁻¹, e.g. 1×10⁻² s⁻¹ to 1×10⁻⁶ s⁻¹,e.g. 5×10⁻³ s⁻¹ to 1×10⁻⁵ s⁻¹, for example 5×10⁻¹ s⁻¹ or less, e.g.1×10⁻² s⁻¹ or less, e.g. 1×10⁻³ s⁻¹ or less, e.g. 1×10⁻⁴ s⁻¹ or less,e.g. 1×10⁻⁵ s¹ or less, and e.g. 1×10⁻⁶ s⁻¹ or less as determined bysurface plasmon resonance.

In certain embodiments, the polypeptide, dAb or antagonist specificallybinds VEGF, eg, human VEGF, with a K_(on) of 1×10⁻³ M⁻¹s⁻¹ to 1×10⁻⁷M⁻¹s⁻¹ or 1×10⁻³ M⁻¹ s⁻¹ to 1×10⁻⁶ M⁻¹s⁻¹ or about 1×10⁻⁴ M⁻¹s⁻¹ orabout 1×10⁻⁵ M⁻¹ s⁻¹. In one embodiment, the polypeptide, dAb orantagonist specifically binds VEGF, eg, human VEGF, and dissociates fromhuman VEGF with a dissociation constant (K_(D)) and a K_(off) as definedin this paragraph. In one embodiment, the polypeptide, dAb or antagonistspecifically binds VEGF, eg, human VEGF, and dissociates from human VEGFwith a dissociation constant (K_(D)) and a K_(on) as defined in thisparagraph. In some embodiments, the polypeptide or dAb specificallybinds VEGF (eg, human VEGF) with a K_(D) and/or K_(off) and/or K_(on) asrecited in this paragraph and comprises an amino acid sequence that isat least or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of a dAb with theamino acid sequence of DOM15-26-593.

The polypeptide, dAb or antagonist can be expressed in E. coli or inPichia species (e.g., P. pastoris). In one embodiment, the ligand or dAbmonomer is secreted in a quantity of at least about 0.5 mg/L whenexpressed in E. coli or in Pichia species (e.g., P. pastoris). Although,the ligands and dAb monomers described herein can be secretable whenexpressed in E. coli or in Pichia species (e.g., P. pastoris), they canbe produced using any suitable method, such as synthetic chemicalmethods or biological production methods that do not employ E. coli orPichia species.

In some embodiments, the polypeptide, dAb or antagonist does notcomprise a Camelid immunoglobulin variable domain, or one or moreframework amino acids that are unique to immunoglobulin variable domainsencoded by Camelid germline antibody gene segments, eg at position 108,37, 44, 45 and/or 47.

Antagonists of VEGF according to the invention can be monovalent ormultivalent. In some embodiments, the antagonist is monovalent andcontains one binding site that interacts with VEGF, the binding siteprovided by a polypeptide or dAb of the invention. Monovalentantagonists bind one VEGF and may not induce cross-linking or clusteringof VEGF on the surface of cells which can lead to activation of thereceptor and signal transduction.

In other embodiments, the antagonist of VEGF is multivalent. Multivalentantagonists of VEGF can contain two or more copies of a particularbinding site for VEGF or contain two or more different binding sitesthat bind VEGF, at least one of the binding sites being provided by apolypeptide or dAb of the invention. For example, as described hereinthe antagonist of VEGF can be a dimer, trimer or multimer comprising twoor more copies of a particular polypeptide or dAb of the invention thatbinds VEGF, or two or more different polypeptides or dAbs of theinvention that bind VEGF. In certain embodiments, the multivalentantagonist of VEGF contains two or more binding sites for a desiredepitope or domain of VEGF.

Some ligands (and antagonists) may have utility as diagnostic agents,because they can be used to bind and detect, quantify or measure VEGF ina sample. Accordingly, an accurate determination of whether or how muchVEGF is in the sample can be made.

In other embodiments, the polypeptide, dAb or antagonist specificallybinds VEGF with a K_(D) described herein and inhibits tumour growth in astandard murine xenograft model (e.g., inhibits tumour growth by atleast about 10%, as compared with a suitable control). In oneembodiment, the polypeptide, dAb or antagonist inhibits tumour growth byat least about 10% or by at least about 25%, or by at least about 50%,as compared to a suitable control in a standard murine xenograft modelwhen administered at about 1 mg/kg or more, for example about 5 or 10mg/kg.

In other embodiments, the polypeptide, dAb or antagonist binds VEGF andantagonizes the activity of the VEGF in a standard cell assay with anND₅₀ of ≦100 nM.

In certain embodiments, the polypeptide, dAb or antagonist of theinvention are efficacious in animal models of inflammatory diseases suchas those described in WO 2006038027 and WO 2006059108 and WO 2007049017when an effective amount is administered. Generally an effective amountis about 1 mg/kg to about 10 mg/kg (e.g., about 1 mg/kg, about 2 mg/kg,about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg). The models ofchronic inflammatory disease are recognized by those skilled in the artas being predictive of therapeutic efficacy in humans.

Generally, the present ligands (e.g., antagonists) will be utilised inpurified form together with pharmacologically appropriate carriers.Typically, these carriers include aqueous or alcoholic/aqueoussolutions, emulsions or suspensions, any including saline and/orbuffered media. Parenteral vehicles include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.Suitable physiologically-acceptable adjuvants, if necessary to keep apolypeptide complex in suspension, may be chosen from thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition). A variety ofsuitable formulations can be used, including extended releaseformulations.

The ligands (e.g., antagonists) of the present invention may be used asseparately administered compositions or in conjunction with otheragents. These can include various immunotherapeutic drugs, such ascylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins.Pharmaceutical compositions can include “cocktails” of various cytotoxicor other agents in conjunction with the ligands of the presentinvention, or even combinations of ligands according to the presentinvention having different specificities, such as ligands selected usingdifferent target antigens or epitopes, whether or not they are pooledprior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, including without limitationimmunotherapy, the selected ligands thereof of the invention can beadministered to any patient in accordance with standard techniques.

The administration can be by any appropriate mode, includingparenterally, intravenously, intramuscularly, intraperitoneally,transdermally, via the pulmonary route, or also, appropriately, bydirect infusion with a catheter. The dosage and frequency ofadministration will depend on the age, sex and condition of the patient,concurrent administration of other drugs, counterindications and otherparameters to be taken into account by the clinician. Administration canbe local (e.g., local delivery to the lung by pulmonary administration,e.g., intranasal administration) or systemic as indicated.

The ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins andart-known lyophilisation and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilisationand reconstitution can lead to varying degrees of antibody activity loss(e.g. with conventional immunoglobulins, IgM antibodies tend to havegreater activity loss than IgG antibodies) and that use levels may haveto be adjusted upward to compensate.

The compositions containing the present ligands (e.g., antagonists) or acocktail thereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of ligand, e.g. dAb or antagonist perkilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being morecommonly used. For prophylactic applications, compositions containingthe present ligands or cocktails thereof may also be administered insimilar or slightly lower dosages, to prevent, inhibit or delay onset ofdisease (e.g., to sustain remission or quiescence, or to prevent acutephase). The skilled clinician will be able to determine the appropriatedosing interval to treat, suppress or prevent disease. When an ligand ofVEGF (e.g., antagonist) is administered to treat, suppress or preventdisease, it can be administered up to four times per day, twice weekly,once weekly, once every two weeks, once a month, or once every twomonths, at a dose off, for example, about 10 μg/kg to about 80 mg/kg,about 100 μg/kg to about 80 mg/kg, about 1 mg/kg to about 80 mg/kg,about 1 mg/kg to about 70 mg/kg, about 1 mg/kg to about 60 mg/kg, about1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 40 mg/kg, about 1mg/kg to about 30 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kgto about 10 mg/kg, about 10 μg/kg to about 10 mg/kg, about 10 μg/kg toabout 5 mg/kg, about 10 μg/kg to about 2.5 mg/kg, about 1 mg/kg, about 2mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg. In particularembodiments, the ligand of VEGF (e.g., antagonist) is administered totreat, suppress or prevent disease once every two weeks or once a monthat a dose of about 10 μg/kg to about 10 mg/kg (e.g., about 10 μg/kg,about 100 μg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about9 mg/kg or about 10 mg/kg.)

Treatment or therapy performed using the compositions described hereinis considered “effective” if one or more symptoms are reduced (e.g., byat least 10% or at least one point on a clinical assessment scale),relative to such symptoms present before treatment, or relative to suchsymptoms in an individual (human or model animal) not treated with suchcomposition or other suitable control. Symptoms will obviously varydepending upon the disease or disorder targeted, but can be measured byan ordinarily skilled clinician or technician. Such symptoms can bemeasured, for example, by monitoring the level of one or morebiochemical indicators of the disease or disorder (e.g., levels of anenzyme or metabolite correlated with the disease, affected cell numbers,etc.), by monitoring physical manifestations (e.g., inflammation, tumorsize, etc.), or by an accepted clinical assessment scale.

Similarly, prophylaxis performed using a composition as described hereinis “effective” if the onset or severity of one or more symptoms isdelayed, reduced or abolished relative to such symptoms in a similarindividual (human or animal model) not treated with the composition.

A composition containing a ligand (e.g., antagonist) or cocktail thereofaccording to the present invention may be utilised in prophylactic andtherapeutic settings to aid in the alteration, inactivation, killing orremoval of a select target cell population in a mammal. In addition, theselected repertoires of polypeptides described herein may be usedextracorporeally or in vitro selectively to kill, deplete or otherwiseeffectively remove a target cell population from a heterogeneouscollection of cells. Blood from a mammal may be combinedextracorporeally with the ligands whereby the undesired cells are killedor otherwise removed from the blood for return to the mammal inaccordance with standard techniques.

A composition containing an ligand (e.g., antagonist) according to thepresent invention may be utilised in prophylactic and therapeuticsettings to aid in the alteration, inactivation, killing or removal of aselect target cell population in a mammal.

The ligands (e.g., anti-VEGF antagonists, dAb monomers) can beadministered and or formulated together with one or more additionaltherapeutic or active agents. When a ligand (eg, a dAb) is administeredwith an additional therapeutic agent, the ligand can be administeredbefore, simultaneously with or subsequent to administration of theadditional agent. Generally, the ligand and additional agent areadministered in a manner that provides an overlap of therapeutic effect.

In one embodiment, the invention is a method for treating, suppressingor preventing disease, selected from for example Cancer (e.g. a solidtumour), inflammatory disease, autoimmune disease, vascularproliferative disease (e.g.AMD (age related macular degeneration))comprising administering to a mammal in need thereof atherapeutically-effective dose or amount of a polypeptide, dAb whichbinds to VEGF or antagonist of VEGF according to the invention.

The invention provides a method for treating, suppressing or preventingpulmonary diseases. Thus, in another embodiment, the invention is amethod for treating, suppressing or preventing a pulmonary disease(e.g., lung cancer) comprising administering to a mammal in need thereofa therapeutically-effective dose or amount of a polypeptide, dAb orantagonist of VEGF according to the invention.

In particular embodiments, an antagonist of VEGF is administered viapulmonary delivery, such as by inhalation (e.g., intrabronchial,intranasal or oral inhalation, intranasal drops) or by systemic delivery(e.g., parenteral, intravenous, intramuscular, intraperitoneal,subcutaneous).

In a further aspect of the invention, there is provided a compositioncomprising a a polypeptide, dAb or antagonist of VEGF according to theinvention and a pharmaceutically acceptable carrier, diluent orexcipient.

Moreover, the present invention provides a method for the treatment ofdisease using a polypeptide, dAb or antagonist of VEGF or a compositionaccording to the present invention. In an embodiment the disease isCancer (e.g. a solid tumour), or an inflammatory disease, eg rheumatoidarthritis, or an autoimmune disease, or a vascular proliferative diseasesuch as AMD (Age Related Macular Degeneration).

Formats

Increased half-life is useful in in vivo applications ofimmunoglobulins, especially antibodies and most especially antibodyfragments of small size. Such fragments (Fvs, disulphide bonded Fvs,Fabs, scFvs, dAbs) suffer from rapid clearance from the body; thus,whilst they are able to reach most parts of the body rapidly, and arequick to produce and easier to handle, their in vivo applications havebeen limited by their only brief persistence in vivo. One embodiment ofthe invention solves this problem by providing increased half-life ofthe ligands in vivo and consequently longer persistence times in thebody of the functional activity of the ligand.

Methods for pharmacokinetic analysis and determination of ligandhalf-life will be familiar to those skilled in the art. Details may befound in Kenneth, A et al: Chemical Stability of Pharmaceuticals: AHandbook for Pharmacists and in Peters et al, Pharmacokinetc analysis: APractical Approach (1996). Reference is also made to “Pharmacokinetics”,M Gibaldi & D Perron, published by Marcel Dekker, 2^(nd) Rev. ex edition(1982), which describes pharmacokinetic parameters such as t alpha and tbeta half lives and area under the curve (AUC).

Half lives (t½ alpha and t½ beta) and AUC can be determined from a curveof serum concentration of ligand against time. The WinNonlin analysispackage (available from Pharsight Corp., Mountain View, Calif. 94040,USA) can be used, for example, to model the curve. In a first phase (thealpha phase) the ligand is undergoing mainly distribution in thepatient, with some elimination. A second phase (beta phase) is theterminal phase when the ligand has been distributed and the serumconcentration is decreasing as the ligand is cleared from the patient.The t alpha half life is the half life of the first phase and the t betahalf life is the half life of the second phase. Thus, in one embodiment,the present invention provides a ligand or a composition comprising aligand according to the invention having a tα half-life in the range of15 minutes or more. In one embodiment, the lower end of the range is 30minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, oralternatively, a ligand or composition according to the invention willhave a tα half life in the range of up to and including 12 hours. In oneembodiment, the upper end of the range is 11, 10, 9, 8, 7, 6 or 5 hours.An example of a suitable range is 1 to 6 hours, 2 to 5 hours or 3 to 4hours.

In one embodiment, the present invention provides a ligand (polypeptide,dAb or antagonist) or a composition comprising a ligand according to theinvention having a tβ half-life in the range of 30 minutes or more. Inone embodiment, the lower end of the range is 45 minutes, 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours,or 12 hours. In addition, or alternatively, a ligand or compositionaccording to the invention has a tβ half-life in the range of up to andincluding 21 days. In one embodiment, the upper end of the range is 12hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20 days. Inone embodiment a ligand or composition according to the invention willhave a tβ half life in the range 12 to 60 hours. In a furtherembodiment, it will be in the range 12 to 48 hours. In a furtherembodiment still, it will be in the range 12 to 26 hours.

In addition, or alternatively to the above criteria, the presentinvention provides a ligand or a composition comprising a ligandaccording to the invention having an AUC value (area under the curve) inthe range of 1 mg·min/ml or more. In one embodiment, the lower end ofthe range is 5, 10, 15, 20, 30, 100, 200 or 300 mg·min/ml. In addition,or alternatively, a ligand or composition according to the invention hasan AUC in the range of up to 600 mg·min/ml. In one embodiment, the upperend of the range is 500, 400, 300, 200, 150, 100, 75 or 50 mg·min/ml. Inone embodiment a ligand according to the invention will have a AUC inthe range selected from the group consisting of the following: 15 to 150mg·min/ml, 15 to 100 mg·min/ml, 15 to 75 mg·min/ml, and 15 to 50mg·min/ml.

Polypeptides and dAbs of the invention and antagonists comprising thesecan be formatted to have a larger hydrodynamic size, for example, byattachment of a PEG group, serum albumin, transferrin, transferrinreceptor or at least the transferrin-binding portion thereof, anantibody Fc region, or by conjugation to an antibody domain. Forexample, polypeptides dAbs and antagonists formatted as a largerantigen-binding fragment of an antibody or as an antibody (e.g.,formatted as a Fab, Fab′, F(ab)₂, F(ab′)₂, IgG, scFv).

Hydrodynamic size of the ligands (e.g., dAb monomers and multimers) ofthe invention may be determined using methods which are well known inthe art. For example, gel filtration chromatography may be used todetermine the hydrodynamic size of a ligand. Suitable gel filtrationmatrices for determining the hydrodynamic sizes of ligands, such ascross-linked agarose matrices, are well known and readily available.

The size of a ligand format (e.g., the size of a PEG moiety attached toa dAb monomer), can be varied depending on the desired application. Forexample, where ligand is intended to leave the circulation and enterinto peripheral tissues, it is desirable to keep the hydrodynamic sizeof the ligand low to facilitate extravazation from the blood stream.Alternatively, where it is desired to have the ligand remain in thesystemic circulation for a longer period of time the size of the ligandcan be increased, for example by formatting as an Ig like protein.

Half-Life Extension by Targeting an Antigen or Epitope that IncreasesHalf-Live In Vivo

The hydrodynaminc size of a ligand and its serum half-life can also beincreased by conjugating or associating a VEGF binding polypeptide, dAbor antagonist of the invention to a binding domain (e.g., antibody orantibody fragment) that binds an antigen or epitope that increaseshalf-live in vivo, as described herein. For example, the VEGF bindingagent (e.g., polypeptide) can be conjugated or linked to an anti-serumalbumin or anti-neonatal Fc receptor antibody or antibody fragment, egan anti-SA or anti-neonatal Fc receptor dAb, Fab, Fab′ or scFv, or to ananti-SA affibody or anti-neonatal Fc receptor Affibody or an anti-SAavimer, or an anti-SA binding domain which comprises a scaffold selectedfrom, but preferably not limited to, the group consisting of CTLA-4,lipocallin, SpA, an affibody, an avimer, GroEl and fibronectin (seePCT/GB2008/000453 filed 8 Feb. 2008 for disclosure of these bindingdomain, which domains and their sequences are incorporated herein byreference and form part of the disclosure of the present text).Conjugating refers to a composition comprising polypeptide, dAb orantagonist of the invention that is bonded (covalently or noncovalently)to a binding domain that binds serum albumin.

Suitable polypeptides that enhance serum half-life in vivo include, forexample, transferrin receptor specific ligand-neuropharmaceutical agentfusion proteins (see U.S. Pat. No. 5,977,307, the teachings of which areincorporated herein by reference), brain capillary endothelial cellreceptor, transferrin, transferrin receptor (e.g., soluble transferrinreceptor), insulin, insulin-like growth factor 1 (IGF 1) receptor,insulin-like growth factor 2 (IGF 2) receptor, insulin receptor, bloodcoagulation factor X, α1-antitrypsin and HNF 1α. Suitable polypeptidesthat enhance serum half-life also include alpha-1 glycoprotein(orosomucoid; AAG), alpha-1 antichymotrypsin (ACT), alpha-1microglobulin (protein HC; AIM), antithrombin III (AT III),apolipoprotein A-1 (Apo A-1), apolipoprotein B (Apo B), ceruloplasmin(Cp), complement component C3 (C3), complement component C4 (C4), C1esterase inhibitor (C1 INH), C-reactive protein (CRP), ferritin (FER),hemopexin (HPX), lipoprotein(a) (Lp(a)), mannose-binding protein (MBP),myoglobin (Myo), prealbumin (transthyretin; PAL), retinol-bindingprotein (RBP), and rheumatoid factor (RF).

Suitable proteins from the extracellular matrix include, for example,collagens, laminins, integrins and fibronectin. Collagens are the majorproteins of the extracellular matrix. About 15 types of collagenmolecules are currently known, found in different parts of the body,e.g. type I collagen (accounting for 90% of body collagen) found inbone, skin, tendon, ligaments, cornea, internal organs or type IIcollagen found in cartilage, vertebral disc, notochord, and vitreoushumor of the eye.

Suitable proteins from the blood include, for example, plasma proteins(e.g., fibrin, α-2 macroglobulin, serum albumin, fibrinogen (e.g.,fibrinogen A, fibrinogen B), serum amyloid protein A, haptoglobin,profilin, ubiquitin, uteroglobulin and β-2-microglobulin), enzymes andenzyme inhibitors (e.g., plasminogen, lysozyme, cystatin C,alpha-1-antitrypsin and pancreatic trypsin inhibitor), proteins of theimmune system, such as immunoglobulin proteins (e.g., IgA, IgD, IgE,IgG, IgM, immunoglobulin light chains (kappa/lambda)), transportproteins (e.g., retinol binding protein, α-1 microglobulin), defensins(e.g., beta-defensin 1, neutrophil defensin 1, neutrophil defensin 2 andneutrophil defensin 3) and the like.

Suitable proteins found at the blood brain barrier or in neural tissueinclude, for example, melanocortin receptor, myelin, ascorbatetransporter and the like.

Suitable polypeptides that enhance serum half-life in vivo also includeproteins localized to the kidney (e.g., polycystin, type IV collagen,organic anion transporter K1, Heymann's antigen), proteins localized tothe liver (e.g., alcohol dehydrogenase, G250), proteins localized to thelung (e.g., secretory component, which binds IgA), proteins localized tothe heart (e.g., HSP 27, which is associated with dilatedcardiomyopathy), proteins localized to the skin (e.g., keratin), bonespecific proteins such as morphogenic proteins (BMPs), which are asubset of the transforming growth factor β superfamily of proteins thatdemonstrate osteogenic activity (e.g., BMP-2, BMP-4, BMP-5, BMP-6,BMP-7, BMP-8), tumor specific proteins (e.g., trophoblast antigen,herceptin receptor, oestrogen receptor, cathepsins (e.g., cathepsin B,which can be found in liver and spleen)).

Suitable disease-specific proteins include, for example, antigensexpressed only on activated T-cells, including LAG-3 (lymphocyteactivation gene), osteoprotegerin ligand (OPGL; see Nature 402, 304-309(1999)), OX40 (a member of the TNF receptor family, expressed onactivated T cells and specifically up-regulated in human T cell leukemiavirus type-I (HTLV-I)-producing cells; see Immunol. 165 (1):263-70(2000)). Suitable disease-specific proteins also include, for example,metalloproteases (associated with arthritis/cancers) including CG6512Drosophila, human paraplegin, human FtsH, human AFG3L2, murine ftsH; andangiogenic growth factors, including acidic fibroblast growth factor(FGF-1), basic fibroblast growth factor (FGF-2), vascular endothelialgrowth factor/vascular permeability factor (VEGF/VPF), transforminggrowth factor-α (TGF α), tumor necrosis factor-alpha (TNF-α),angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derivedendothelial growth factor (PD-ECGF), placental growth factor (P1GF),midkine platelet-derived growth factor-BB (PDGF), and fractalkine.

Suitable polypeptides that enhance serum half-life in vivo also includestress proteins such as heat shock proteins (HSPs). HSPs are normallyfound intracellularly. When they are found extracellularly, it is anindicator that a cell has died and spilled out its contents. Thisunprogrammed cell death (necrosis) occurs when as a result of trauma,disease or injury, extracellular HSPs trigger a response from the immunesystem. Binding to extracellular HSP can result in localizing thecompositions of the invention to a disease site.

Suitable proteins involved in Fc transport include, for example,Brambell receptor (also known as FcRB). This Fc receptor has twofunctions, both of which are potentially useful for delivery. Thefunctions are (1) transport of IgG from mother to child across theplacenta (2) protection of IgG from degradation thereby prolonging itsserum half-life. It is thought that the receptor recycles IgG fromendosomes. (See, Holliger et al, Nat Biotechnol 15(7):632-6 (1997).)

dAbs that Bind Serum Albumin

The invention in one embodiment provides a polypeptide or antagonist(e.g., dual specific ligand comprising an anti-TNFR1 dAb (a first dAb)that binds to TNFR1 a second dAb that binds serum albumin (SA), thesecond dAb binding SA with a K_(D) as determined by surface plasmonresonance of 1 nM to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 100,200, 300, 400 or 500 μM (i.e., ×10⁻⁹ to 5×10⁻⁴), or 100 nM to 10 μM, or1 to 5 μM or 3 to 70 nM or 10 nM to 1, 2, 3, 4 or 5 μM. For example 30to 70 nM as determined by surface plasmon resonance. In one embodiment,the first dAb (or a dAb monomer) binds SA (e.g., HSA) with a K_(D) asdetermined by surface plasmon resonance of approximately 1, 50, 70, 100,150, 200, 300 nM or 1, 2 or 3 μM. In one embodiment, for a dual specificligand comprising a first anti-SA dAb and a second dAb to VEGF, theaffinity (eg K_(D) and/or K_(off) as measured by surface plasmonresonance, eg using BiaCore) of the second dAb for its target is from 1to 100000 times (eg, 100 to 100000, or 1000 to 100000, or 10000 to100000 times) the affinity of the first dAb for SA. In one embodiment,the serum albumin is human serum albumin (HSA). For example, the firstdAb binds SA with an affinity of approximately 10 μM, while the seconddAb binds its target with an affinity of 100 μM. In one embodiment, theserum albumin is human serum albumin (HSA). In one embodiment, the firstdAb binds SA (eg, HSA) with a K_(D) of approximately 50, for example 70,100, 150 or 200 nM. Details of dual specific ligands are found inWO03002609, WO04003019 and WO04058821.

The ligands of the invention can in one embodiment comprise a dAb thatbinds serum albumin (SA) with a K_(D) as determined by surface plasmonresonance of 1 nM to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 100,200, 300, 400 or 500 μM (i.e., ×10-9 to 5×10-4), or 100 nM to 10 μM, or1 to 5 μM or 3 to 70 nM or 10 nM to 1, 2, 3, 4 or 5 μM. For example 30to 70 nM as determined by surface plasmon resonance. In one embodiment,the first dAb (or a dAb monomer) binds SA (e.g., HSA) with a K_(D) asdetermined by surface plasmon resonance of approximately 1, 50, 70, 100,150, 200, 300 nM or 1, 2 or 3 μM. In one embodiment, the first andsecond dAbs are linked by a linker, for example a linker of from 1 to 4amino acids or from 1 to 3 amino acids, or greater than 3 amino acids orgreater than 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acids. In oneembodiment, a longer linker (greater than 3 amino acids) is used toenhance potency (K_(D) of one or both dAbs in the antagonist).

In particular embodiments of the ligands and antagonists, the dAb bindshuman serum albumin and competes for binding to albumin with a dAbselected from the group consisting of

MSA-16, MSA-26 (See WO04003019 for disclosure of these sequences, whichsequences and their nucleic acid counterpart are incorporated herein byreference and form part of the disclosure of the present text),

DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474), DOM7m-26 (SEQ IDNO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ ID NO: 477), DOM7r-4(SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479), DOM7r-7 (SEQ ID NO: 480),DOM7r-8 (SEQ ID NO: 481), DOM7h-2 (SEQ ID NO: 482), DOM7h-3 (SEQ ID NO:483), DOM7h-4 (SEQ ID NO: 484), DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQID NO: 486), DOM7h-7 (SEQ ID NO: 487), DOM7h-22 (SEQ ID NO: 489),DOM7h-23 (SEQ ID NO: 490), DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ IDNO: 492), DOM7h-26 (SEQ ID NO: 493), DOM7h-21 (SEQ ID NO: 494), DOM7h-27(SEQ ID NO: 495), DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497),DOM7r-14 (SEQ ID NO: 498), DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ IDNO: 500), DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502), DOM7r-19(SEQ ID NO: 503), DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505),DOM7r-22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507), DOM7r-24 (SEQ IDNO: 508), DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510), DOM7r-27(SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512), DOM7r-29 (SEQ ID NO: 513),DOM7r-30 (SEQ ID NO: 514), DOM7r-31 (SEQ ID NO: 515), DOM7r-32 (SEQ IDNO: 516), DOM7r-33 (SEQ ID NO: 517) (See WO2007080392 for disclosure ofthese sequences, which sequences and their nucleic acid counterpart areincorporated herein by reference and form part of the disclosure of thepresent text; the SEQ ID No's in this paragraph are those that appear inWO2007080392),

dAb8 (dAb10), dAb 10, dAb36, dAb7r20 (DOM7r20), dAb7r21 (DOM7r21),dAb7r22 (DOM7r22), dAb7r23 (DOM7r23), dAb7r24 (DOM7r24), dAb7r25(DOM7r25), dAb7r26 (DOM7r26), dAb7r27 (DOM7r27), dAb7r28 (DOM7r28),dAb7r29 (DOM7r29), dAb7r29 (DOM7r29), dAb7r31 (DOM7r31), dAb7r32(DOM7r32), dAb7r33 (DOM7r33), dAb7r33 (DOM7r33), dAb7h22 (DOM7h22),dAb7h23 (DOM7h23), dAb7h24 (DOM7h24), dAb7h25 (DOM7h25), dAb7h26(DOM7h26), dAb7h27 (DOM7h27), dAb7h30 (DOM7h30), dAb7h31 (DOM7h31), dAb2(dAbs 4, 7, 41), dAb4, dAb7, dAb11, dAb12 (dAb7 m12), dAb13 (dAb 15),dAb15, dAb16 (dAb21, dAb7 m16), dAb17, dAb18, dAb19, dAb21, dAb22,dAb23, dAb24, dAb25 (dAb26, dAb7 m26), dAb27, dAb30 (dAb35), dAb31,dAb33, dAb34, dAb35, dAb38 (dAb54), dAb41, dAb46 (dAbs 47, 52 and 56),dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, dAb7 m12, dAb7 m16, dAb7 m26,dAb7r1 (DOM7r1), dAb7r3 (DOM7r3), dAb7r4 (DOM7r4), dAb7r5 (DOM7r5),dAb7r7 (DOM7r7), dAb7r8 (DOM7r8), dAb7r13 (DOM7r13), dAb7r14 (DOM7r14),dAb7r15 (DOM7r15), dAb7r16 (DOM7r16), dAb7r17 (DOM7r17), dAb7r18(DOM7r18), dAb7r19 (DOM7r19), dAb7h1 (DOM7h1), dAb7h2 (DOM7h2), dAb7h6(DOM7h6), dAb7h7 (DOM7h7), dAb7h8 (DOM7h8), dAb7h9 (DOM7h9), dAb7h10(DOM7h10), dAb7h11 (DOM7h11), dAb7h12 (DOM7h12), dAb7h13 (DOM7h13),dAb7h14 (DOM7h14), dAb7p1 (DOM7p1), and dAb7p2 (DOM7p2) (seePCT/GB2008/000453 filed 8 Feb. 2008 for disclosure of these sequences,which sequences and their nucleic acid counterpart are incorporatedherein by reference and form part of the disclosure of the presenttext). Alternative names are shown in brackets after the dAb, e.g. dAb8has an alternative name which is dAb10 i.e. dAb8 (dAb10). Thesesequences are also set out in FIGS. 51 a and b.

In certain embodiments, the dAb binds human serum albumin and comprisesan amino acid sequence that has at least about 80%, or at least about85%, or at least about 90%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98%, or at least about 99%amino acid sequence identity with the amino acid sequence of a dAbselected from the group consisting of

MSA-16, MSA-26,

DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474), DOM7m-26 (SEQ IDNO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ ID NO: 477), DOM7r-4(SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479), DOM7r-7 (SEQ ID NO: 480),DOM7r-8 (SEQ ID NO: 481), DOM7h-2 (SEQ ID NO: 482), DOM7h-3 (SEQ ID NO:483), DOM7h-4 (SEQ ID NO: 484), DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQID NO: 486), DOM7h-7 (SEQ ID NO: 487), DOM7h-22 (SEQ ID NO: 489),DOM7h-23 (SEQ ID NO: 490), DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ IDNO: 492), DOM7h-26 (SEQ ID NO: 493), DOM7h-21 (SEQ ID NO: 494), DOM7h-27(SEQ ID NO: 495), DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497),DOM7r-14 (SEQ ID NO: 498), DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ IDNO: 500), DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502), DOM7r-19(SEQ ID NO: 503), DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505),DOM7r-22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507), DOM7r-24 (SEQ IDNO: 508), DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510), DOM7r-27(SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512), DOM7r-29 (SEQ ID NO: 513),DOM7r-30 (SEQ ID NO: 514), DOM7r-31 (SEQ ID NO: 515), DOM7r-32 (SEQ IDNO: 516), DOM7r-33 (SEQ ID NO: 517) (the SEQ ID No's in this paragraphare those that appear in WO2007080392),

dAb8, dAb 10, dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24,dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32,dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16,dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27,dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52,dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3,dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2.

For example, the dAb that binds human serum albumin can comprise anamino acid sequence that has at least about 90%, or at least about 95%,or at least about 96%, or at least about 97%, or at least about 98%, orat least about 99% amino acid sequence identity with DOM7h-2 (SEQ IDNO:482), DOM7h-3 (SEQ ID NO:483), DOM7h-4 (SEQ ID NO:484), DOM7h-6 (SEQID NO:485), DOM7h-1 (SEQ ID NO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8(SEQ ID NO:496), DOM7r-13 (SEQ ID NO:497), DOM7r-14 (SEQ ID NO:498),DOM7h-22 (SEQ ID NO:489), DOM7h-23 (SEQ ID NO:490), DOM7h-24 (SEQ IDNO:491), DOM7h-25 (SEQ ID NO:492), DOM7h-26 (SEQ ID NO:493), DOM7h-21(SEQ ID NO:494), DOM7h-27 (SEQ ID NO:495) (the SEQ ID No's in thisparagraph are those that appear in WO2007080392),

dAb8, dAb 10, dAb36, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26,dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15,dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26,dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47,dAb52, dAb53, dAb54, dAb55, dAb56, dAb7h1, dAb7h2, dAb7h6, dAb7h7,dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13 and dAb7h14

In certain embodiments, the dAb binds human serum albumin and comprisesan amino acid sequence that has at least about 80%, or at least about85%, or at least about 90%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98%, or at least about 99%amino acid sequence identity with the amino acid sequence of a dAbselected from the group consisting of

DOM7h-2 (SEQ ID NO:482), DOM7h-6 (SEQ ID NO:485), DOM7h-1 (SEQ IDNO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8 (SEQ ID NO:496), DOM7h-22 (SEQID NO:489), DOM7h-23 (SEQ ID NO:490), DOM7h-24 (SEQ ID NO:491), DOM7h-25(SEQ ID NO:492), DOM7h-26 (SEQ ID NO:493), DOM7h-21 (SEQ ID NO:494),DOM7h-27 (SEQ ID NO:495) (the SEQ ID No's in this paragraph are thosethat appear in WO2007080392),

dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30,dAb7h31, dAb2, dAb4, dAb7, dAb38, dAb41, dAb7h1, dAb7h2, dAb7h6, dAb7h7,dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13 and dAb7h14.

In more particular embodiments, the dAb is a V_(κ) dAb that binds humanserum albumin and has an amino acid sequence selected from the groupconsisting of

DOM7h-2 (SEQ ID NO:482), DOM7h-6 (SEQ ID NO:485), DOM7h-1 (SEQ IDNO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8 (SEQ ID NO:496) (the SEQ IDNo's in this paragraph are those that appear in WO2007080392),

dAb2, dAb4, dAb7, dAb38, dAb41, dAb54, dAb7h1, dAb7h2, dAb7h6, dAb7h7,dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13 and dAb7h14,

In more particular embodiments, the dAb is a V_(H) dAb that binds humanserum albumin and has an amino acid sequence selected from dAb7h30 anddAb7h31.

In more particular embodiments, the dAb is dAb7h11 or dAb7h14.

In other embodiments, the dAb, ligand or antagonist binds human serumalbumin and comprises one, two or three of the CDRs of any of theforegoing amino acid sequences, eg one, two or three of the CDRs ofdAb7h11 or dAb7h14.

Suitable Camelid V_(HH) that bind serum albumin include those disclosedin WO 2004/041862 (Ablynx N.V.) and in WO2007080392 (which V_(HH)sequences and their nucleic acid counterpart are incorporated herein byreference and form part of the disclosure of the present text), such asSequence A (SEQ ID NO:518), Sequence B (SEQ ID NO:519), Sequence C (SEQID NO:520), Sequence D (SEQ ID NO:521), Sequence E (SEQ ID NO:522),Sequence F (SEQ ID NO:523), Sequence G (SEQ ID NO:524), Sequence H (SEQID NO:525), Sequence I (SEQ ID NO:526), Sequence J (SEQ ID NO:527),Sequence K (SEQ ID NO:528), Sequence L (SEQ ID NO:529), Sequence M (SEQID NO:530), Sequence N (SEQ ID NO:531), Sequence O (SEQ ID NO:532),Sequence P (SEQ ID NO:533), Sequence Q (SEQ ID NO:534), these sequencenumbers corresponding to those cited in WO2007080392 or WO 2004/041862(Ablynx N.V.). In certain embodiments, the Camelid V_(HH) binds humanserum albumin and comprises an amino acid sequence that has at leastabout 80%, or at least about 85%, or at least about 90%, or at leastabout 95%, or at least about 96%, or at least about 97%, or at leastabout 98%, or at least about 99% amino acid sequence identity with ALB1disclosed in WO2007080392 or with any one of SEQ ID NOS:518-534, thesesequence numbers corresponding to those cited in WO2007080392 or WO2004/041862.

In some embodiments, the ligand or antagonist comprises an anti-serumalbumin dAb that competes with any anti-serum albumin dAb disclosedherein for binding to serum albumin (e.g., human serum albumin). In analternative embodiment, the antagonist or ligand comprises a bindingmoiety specific for VEGF (eg, human VEGF), wherein the moiety comprisesnon-immunoglobulin sequences as described in co-pending applicationPCT/GB2008/000453 filed 8 Feb. 2008, the disclosure of these bindingmoieties, their methods of production and selection (eg, from diverselibraries) and their sequences are incorporated herein by reference aspart of the disclosure of the present text)

Conjugation to a Half-Life Extending Moiety (Eg, Albumin)

In one embodiment, a (one or more) half-life extending moiety (eg,albumin, transferrin and fragments and analogues thereof) is conjugatedor associated with the VEGF-binding polypeptide, dAb or antagonist ofthe invention. Examples of suitable albumin, albumin fragments oralbumin variants for use in a VEGF-binding format are described in WO2005077042, which disclosure is incorporated herein by reference andforms part of the disclosure of the present text. In particular, thefollowing albumin, albumin fragments or albumin variants can be used inthe present invention:

-   -   SEQ ID NO:1 (as disclosed in WO 2005077042, this sequence being        explicitly incorporated into the present disclosure by        reference);    -   Albumin fragment or variant comprising or consisting of amino        acids 1-387 of SEQ ID NO:1 in WO 2005077042;    -   Albumin, or fragment or variant thereof, comprising an amino        acid sequence selected from the group consisting of: (a) amino        acids 54 to 61 of SEQ ID NO:1 in WO 2005077042; (b) amino acids        76 to 89 of SEQ ID NO:1 in WO 2005077042; (c) amino acids 92 to        100 of SEQ ID NO:1 in WO 2005077042; (d) amino acids 170 to 176        of SEQ ID NO:1 in WO 2005077042; (e) amino acids 247 to 252 of        SEQ ID NO:1 in WO 2005077042; (f) amino acids 266 to 277 of SEQ        ID NO:1 in WO 2005077042; (g) amino acids 280 to 288 of SEQ ID        NO:1 in WO 2005077042; (h) amino acids 362 to 368 of SEQ ID NO:1        in WO 2005077042; (i) amino acids 439 to 447 of SEQ ID NO:1 in        WO 2005077042 (j) amino acids 462 to 475 of SEQ ID NO:1 in WO        2005077042; (k) amino acids 478 to 486 of SEQ ID NO:1 in WO        2005077042; and (l) amino acids 560 to 566 of SEQ ID NO:1 in WO        2005077042.

Further examples of suitable albumin, fragments and analogs for use in aVEGF binding format are described in WO 03076567, which disclosure isincorporated herein by reference and which forms part of the disclosureof the present text. In particular, the following albumin, fragments orvariants can be used in the present invention:

-   -   Human serum albumin as described in WO 03076567, eg, in FIG. 3        (this sequence information being explicitly incorporated into        the present disclosure by reference);    -   Human serum albumin (HA) consisting of a single non-glycosylated        polypeptide chain of 585 amino acids with a formula molecular        weight of 66,500 (See, Meloun, et al., FEBS Letters 58:136        (1975); Behrens, et al., Fed. Proc. 34:591 (1975); Lawn, et al.,        Nucleic Acids Research 9:6102-6114 (1981); Minghetti, et al., J.        Biol. Chem. 261:6747 (1986));    -   A polymorphic variant or analog or fragment of albumin as        described in Weitkamp, et al., Ann. Hum. Genet. 37:219 (1973);    -   An albumin fragment or variant as described in EP 322094, eg,        HA(1-373, HA(1-388), HA(1-389), HA(1-369), and HA(1-419) and        fragments between 1-369 and 1-419;    -   An albumin fragment or variant as described in EP 399666, eg,        HA(1-177) and HA(1-200) and fragments between HA(1-X), where X        is any number from 178 to 199.

Where a (one or more) half-life extending moiety (eg, albumin,transferrin and fragments and analogues thereof) is used to format theVEGF-binding polypeptides, dAbs and antagonists of the invention, it canbe conjugated using any suitable method, such as, by direct fusion tothe VEGF-binding moiety (eg, anti-VEGF dAb), for example by using asingle nucleotide construct that encodes a fusion protein, wherein thefusion protein is encoded as a single polypeptide chain with thehalf-life extending moiety located N- or C-terminally to the VEGFbinding moiety. Alternatively, conjugation can be achieved by using apeptide linker between moieties, eg, a peptide linker as described in WO03076567 or WO 2004003019 (these linker disclosures being incorporatedby reference in the present disclosure to provide examples for use inthe present invention). Typically, a polypeptide that enhances serumhalf-life in vivo is a polypeptide which occurs naturally in vivo andwhich resists degradation or removal by endogenous mechanisms whichremove unwanted material from the organism (e.g., human). For example, apolypeptide that enhances serum half-life in vivo can be selected fromproteins from the extracellular matrix, proteins found in blood,proteins found at the blood brain barrier or in neural tissue, proteinslocalized to the kidney, liver, lung, heart, skin or bone, stressproteins, disease-specific proteins, or proteins involved in Fctransport.

In embodiments of the invention described throughout this disclosure,instead of the use of an anti-VEGF “dAb” in an antagonist or ligand ofthe invention, it is contemplated that the skilled addressee can use apolypeptide or domain that comprises one or more or all 3 of the CDRs ofa dAb of the invention that binds VEGF (e.g., CDRs grafted onto asuitable protein scaffold or skeleton, eg an affibody, an SpA scaffold,an LDL receptor class A domain or an EGF domain) The disclosure as awhole is to be construed accordingly to provide disclosure ofantagonists using such domains in place of a dAb. In this respect, seePCT/GB2008/000453 filed 8 Feb. 2008, the disclosure of which isincorporated by reference).

In one embodiment, therefore, an antagonist of the invention comprisesan immunoglobulin single variable domain or domain antibody (dAb) thathas binding specificity for VEGF or the complementarity determiningregions of such a dAb in a suitable format. The antagonist can be apolypeptide that consists of such a dAb, or consists essentially of sucha dAb. The antagonist can be a polypeptide that comprises a dAb (or theCDRs of a dAb) in a suitable format, such as an antibody format (e.g.,IgG-like format, scFv, Fab, Fab′, F(ab′)₂), or a dual specific ligandthat comprises a dAb that binds VEGF and a second dAb that binds anothertarget protein, antigen or epitope (e.g., serum albumin).

Polypeptides, dAbs and antagonists according to the invention can beformatted as a variety of suitable antibody formats that are known inthe art, such as, IgG-like formats, chimeric antibodies, humanizedantibodies, human antibodies, single chain antibodies, bispecificantibodies, antibody heavy chains, antibody light chains, homodimers andheterodimers of antibody heavy chains and/or light chains,antigen-binding fragments of any of the foregoing (e.g., a Fv fragment(e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fab fragment, aFab′ fragment, a F(ab′)₂ fragment), a single variable domain (e.g.,V_(H), V_(L)), a dAb, and modified versions of any of the foregoing(e.g., modified by the covalent attachment of polyalkylene glycol (e.g.,polyethylene glycol, polypropylene glycol, polybutylene glycol) or othersuitable polymer).

In some embodiments, the invention provides a ligand (eg, an anti-VEGFantagonist) that is an IgG-like format. Such formats have theconventional four chain structure of an IgG molecule (2 heavy chains andtwo light chains), in which one or more of the variable regions (V_(H)and or V_(L)) have been replaced with a dAb of the invention. In oneembodiment, each of the variable regions (2 V_(H) regions and 2 V_(L)regions) is replaced with a dAb or single variable domain, at least oneof which is an anti-VEGF dAb according to the invention. The dAb(s) orsingle variable domain(s) that are included in an IgG-like format canhave the same specificity or different specificities. In someembodiments, the IgG-like format is tetravalent and can have one(anti-VEGF only), two (eg, anti-VEGF and anti-SA), three or fourspecificities. For example, the IgG-like format can be monospecific andcomprises 4 dAbs that have the same specificity; bispecific andcomprises 3 dAbs that have the same specificity and another dAb that hasa different specificity; bispecific and comprise two dAbs that have thesame specificity and two dAbs that have a common but differentspecificity; trispecific and comprises first and second dAbs that havethe same specificity, a third dAb with a different specificity and afourth dAb with a different specificity from the first, second and thirddAbs; or tetraspecific and comprise four dAbs that each have a differentspecificity. Antigen-binding fragments of IgG-like formats (e.g., Fab,F(ab′)₂, Fab′, Fv, scF_(V)) can be prepared. In one embodiment, theIgG-like formats or antigen-binding fragments thereof do not crosslinkVEGF, for example, the format may be monovalent for VEGF. If complementactivation and/or antibody dependent cellular cytotoxicity (ADCC)function is desired, the ligand can be an IgG1-like format. If desired,the IgG-like format can comprise a mutated constant region (variant IgGheavy chain constant region) to minimize binding to Fc receptors and/orability to fix complement. (see e.g. Winter et al., GB 2,209,757 B;Morrison et al., WO 89/07142; Morgan et al., WO 94/29351, Dec. 22,1994).

The ligands of the invention (polypeptides, dAbs and antagonists) can beformatted as a fusion protein that contains a first immunoglobulinsingle variable domain that is fused directly to a second immunoglobulinsingle variable domain. If desired such a format can further comprise ahalf-life extending moiety. For example, the ligand can comprise a firstimmunoglobulin single variable domain that is fused directly to a secondimmunoglobulin single variable domain that is fused directly to animmunoglobulin single variable domain that binds serum albumin.

Generally the orientation of the polypeptide domains that have a bindingsite with binding specificity for a target, and whether the ligandcomprises a linker, is a matter of design choice. However, someorientations, with or without linkers, may provide better bindingcharacteristics than other orientations. All orientations (e.g.,dAb1-linker-dAb2; dAb2-linker-dAb1) are encompassed by the invention areligands that contain an orientation that provides desired bindingcharacteristics can be easily identified by screening.

Polypeptides and dAbs according to the invention, including dAbmonomers, dimers and trimers, can be linked to an antibody Fc region,comprising one or both of C_(H)2 and C_(H)3 domains, and optionally ahinge region. For example, vectors encoding ligands linked as a singlenucleotide sequence to an Fc region may be used to prepare suchpolypeptides.

The invention moreover provides dimers, trimers and polymers of theaforementioned dAb monomers e.g. of anti-VEGF dAb monomers.

Codon Optimised Sequences

As described above, embodiments of the invention provide codon optimizednucleotide sequences encoding polypeptides and variable domains of theinvention. As shown in the following illustration, codon optimizedsequences of about 70% identity can be produced that encode for the samevariable domain (in this case the variable domain amino acid sequence isidentical to DOM1h-131-206). In this instance, the sequences wereoptimized for expression by Pichia pastoris (codon optimized sequences1-3) or E. coli (codon optimized sequences 4 and 5).

We performed a calculation taking into account the degeneracy in thegenetic code and maximised the number of nucleotide changes within eachdegenerate codon encoded by the nucleotide sequence of DOM1h-131-206 (asshown below as DOM1h-131-206 WT) and a theoretical nucleotide sequencewhich still encodes a variable domain that is identical toDOM1h-131-206. The calculation revealed that the theoretical sequencewould have only 57% identity to the nucleotide sequence ofDOM1h-131-206.

Codon Optimised Sequence 1

DNA Sequence (SEQ ID NO: 241)

gaggttcaattgttggaatccggtggtggattggttcaacctggtggttctttgagattgtcctgtgctgcttccggttttactttcgctcacgagactatggtttgggttagacaggctccaggtaaaggattggaatgggtttcccacattccaccagatggtcaagatccattctacgctgactccgttaagggaagattcactatctccagagacaactccaagaacactttgtacttgcagatgaactccttgagagctgaggatactgctgtttaccactgtgctttgttgccaaagagaggaccttggtttgattactggggacagggaactttggttactgt ttcttccCorresponding AA Sequence (SEQ ID NO: 242)

evqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvshippdgqdpfyadsvkgrftisrdnskntlylqmnslraedtavyhcallp krgpwfdywgqgtlvtvss

-   -   74.1% nucleotide sequence identity to WT sequence

Codon Optimised Sequence 2DNA Sequence (SEQ ID NO: 243)

gagaaaagagaggttcaattgcttgaatctggaggaggtttggtccagccaggagggtcccttcgactaagttgtgctgccagtgggtttacgtttgctcatgaaactatggtatgggtccgacaggcacctggtaaaggtcttgaatgggtttcacatatccctccagacggtcaagacccattttacgctgattccgtgaaaggcagatttacaatttcacgagataattctaaaaacaccttgtacttacaaatgaactcattgagagctgaggacactgcagtttatcactgcgctttactaccaaaacgtggaccttggtttgattattggggccaaggtacgtt agtgactgttagttctCorresponding AA Sequence (SEQ ID NO: 244)

ekrevqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvshippdgqdpfyadsvkgrftisrdnskntlylqmnslraedtavyhcallpkrgpwfdywgqgtlvtvss

-   -   71.1% nucleotide sequence identity to WT sequence

Codon Optimised Sequence 3DNA Sequence (SEQ ID NO: 245)

gaagtgcagcttcttgaaagtggtggagggctagtgcagccagggggatctttaagattatcatgcgctgccagtggatttacttttgctcacgagacgatggtctgggtgagacaagctcctggaaaaggtttagagtgggtttctcacattccacctgatggtcaagatcctttctacgcagattccgtcaaaggaagatttactatctccagagataatagtaaaaacactttgtacctacagatgaactcacttagagccgaagataccgctgtgtaccactgcgccttgttgccaaagagaggtccttggttcgattactggggtcagggtactctggttacagt ctcatctCorresponding AA Sequence (SEQ ID NO: 247)

evqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvshippdgqdpfyadsvkgrftisrdnskntlylqmnslraedtavyhcallp krgpwfdywgqgtlvtvss

-   -   72.6% nucleotide sequence identity to WT sequence

Codon Optimised Sequence 4DNA Sequence (SEQ ID NO: 247)

gaagtacaactgctggagagcggtggcggcctggttcaaccgggtggttccctgcgcctgtcctgtgcggcatctggtttcaccttcgcacacgaaaccatggtgtgggttcgccaagctccgggcaaaggcctggaatgggtaagccacattcctccagatggccaggacccattctatgcggattccgttaagggtcgctttaccatttctcgtgataactccaaaaacaccctgtacctgcagatgaactccctgcgcgccgaggatactgcggtgtaccattgtgcgctgctgcctaaacgtggcccgtggttcgattactggggtcagggtactctggtcaccgt aagcagcCorresponding AA Sequence (SEQ ID NO: 248)

evqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvshippdgqdpfyadsvkgrftisrdnskntlylqmnslraedtavyhcallp krgpwfdywgqgtlvtvss

-   -   76.5% nucleotide sequence identity to WT sequence

Codon Optimised Sequence 5DNA Sequence (SEQ ID NO: 249)

gaggttcaactgctggaatctggtggtggtctggtacaaccgggtggttccctgcgtctgagctgtgcagcctctggtttcaccttcgctcatgagaccatggtttgggtacgccaggctccgggtaaaggcctggagtgggtaagccatatccctcctgatggtcaggacccgttctatgctgattccgtcaaaggccgttttaccatttctcgtgacaacagcaaaaacactctgtacctgcaaatgaactccctgcgtgcagaagacacggcggtttatcactgtgcactgctgccaaaacgcggcccttggttcgactactggggccagggtactctggtcactgt atcttctCorresponding AA Sequence (SEQ ID NO: 250)

evqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvshippdgqdpfyadsvkgrftisrdnskntlylqmnslraedtavyhcallp krgpwfdywgqgtlvtvss

-   -   78.4% nucleotide sequence identity to WT sequence

EXEMPLIFICATION Example A Lead Selection & Characterisation of DomainAntibodies to Human TNFR1

Domain antibodies generated were derived from phage libraries. Bothsoluble selections and panning to passively absorbed human TNFR1 wereperformed according to the relevant standard methods. Human TNFR1 waspurchased as a soluble recombinant protein either from R&D systems (CatNo 636-R1-025/CF) or Peprotech (Cat no. 310-07) and either used directly(in the case of passive selections) or after biotinylation usingcoupling via primary amines followed by quality control of its activityin a biological assay and analysis of its MW and extent of biotinylationby mass spectrometry. Typically 3 rounds of selection were performedutilising decreasing levels of antigen in every next round.

Outputs from selections were screened by phage ELISA for the presence ofanti-TNFR1 binding clones. DNA was isolated from these phage selectionsand subcloned into a expression vector for expression of soluble dAbfragments. Soluble dAb fragments were expressed in 96-well plates andthe supernantants were used to screen for the presence of anti-TNFR1binding dAbs, either using a direct binding ELISA with anti-c-mycdetection or BIAcore™ using a streptavidin/biotinylated TNFR1 BIAcore™chip and ranked according to off-rates.

The lead molecules, described below, were derived from the parental dAb,designated DOM1h-131 (disclosed in WO2006038027). This molecule wasselected from the phage display library after 3 rounds of selectionsusing 60 nM of biotinylated antigen. Streptavidin or neutravidin coatedDyna beads were alternated as capture reagents in each round ofselection to prevent selection of binders against either streptavidin orneutravidin. The potency of the lead DOM1h-131 at this stage was in thelow micromolar range as determined in the MRC-5 fibroblast/IL-8 releasecell assay. The binding kinetics as determined by BIAcore™ typicallydisplayed fast-on/fast-off rates. E. coli expression levels of thisDOM1h-131 lead molecule, as a C-terminally myc tagged monomer were inthe region of 8 mg/l.

Affinity Maturation of Leads:

DOM1h-131 was taken forward into affinity maturation to generate mutantswith higher potency and improved biophysical characteristics (see FIG. 3for amino acid sequences of DOM1h-131 derived leads). After generationof an error-prone library (average number of 1 amino acid change per dAbsequence, library size 8×10⁷) using an error-prone PCR polymerase(Genemorph II, Stratagene), seven rounds of selection utilising theseerror-prone libraries were performed. This strategy led to the isolationof clone DOM1h-131-8, a molecule where 4 amino acid changes (one inframework 1 (FR1), one in CDR1, one in CDR3 and one in FR4) gave anapproximate 100-fold improvement in potency as measured by the MRC-5cell assay (˜4 nM). In this assay MRC-5 cells were incubated with thetest samples for one hour then TNF-α (200 pg/ml) was added. After anovernight incubation IL-8 release was determined using an IL-8 ABI 8200cellular detection assay (FMAT). A TNF-α dose curve was included in eachexperiment. The concentration of TNF-α used to compete with dAb bindingto TNFR1 (200 pg/ml) was approximately 70% of the maximum TNF-α responsein this assay.

In order to further improve potency, single amino acid positions werediversified by oligo-directed mutagenesis at key positions suggested bythe error-prone lead consensus information. During this process animproved version of the DOM1h-131-8 clone, DOM1h-131-24 (originallynamed DOM1h-131-8-2 prior to correction) was isolated through BIAcore™screening that had a single K94R amino acid mutation (amino acidnumbering according to Kabat) and an RBA potency of 200-300 pM.

Further error-prone libraries based on this lead and the NNS libraryfrom which it was derived were generated and subjected to three roundsof phage selections using heat treatment (for method see Jespers L, etal., Aggregation-resistant domain antibodies selected on phage by heatdenaturation. Nat. Biotechnol. 2004 September; 22(9):1161-5). Duringthis selection, libraries were pooled and clones derived from round twoof the selection yielded dAbs such as DOM1h-131-53 which were consideredto be more heat stable. It was hypothesised that these clones wouldpossess better biophysical characteristics. Some framework mutations inclone DOM1h-131-53 were germlined to generate clone DOM1h-131-83. Thisclone formed the basis for further diversification via oligo-directedindividual CDR mutagenesis either using phage display selection asdescribed above or using the in-vitro compartmentalization technologyusing emulsions. The phage display strategy generated leadsDOM1h-131-117 and DOM1h-131-151. The in-vitro compartmentalizationtechnology generated DOM1h-131-511.

At this stage these three leads were compared in biophysical andbiological assays and DOM1h-131-511 was the molecule with the bestproperties. Furthermore these molecules were tested for their resistanceto proteolytic cleavage in the presence of trypsin or leucozyme.Leucozyme consists of pooled sputum from patients with cystic fibrosisand contains high levels of elastase and other proteases and was used asa surrogate for in vivo conditions in lung diseases. This data indicatedthat all three leads DOM1h-131-117, DOM1h-131-151 and DOM1h-131-511 wererapidly degraded in presence of trypsin or leucozyme. This findingraised concerns about the in vivo persistence of DOM1h-131-511 when inthe patient and a strategy was developed to select for improvedresistance to trypsin. It was hypothesised that such improved trypsinresistance could have a beneficial effect on other biophysicalproperties of the molecule. Essentially the standard phage selectionmethod was modified to allow for selection in the presence of proteasesprior to selection on antigen. To this end a new phage vector wasengineered in which the c-myc tag was deleted to allow selections in thepresence of trypsin without cleaving the displayed dAb off the phage.DOM1h-131-511 based error-prone libraries were generated and cloned inthe pDOM33 vector (see FIG. 50 for pDOM33 vector map). Phage stocksgenerated from this library were pre-treated with either 1 mg/ml or 100μg/ml trypsin at 37° C. for 24 hours, subsequently protease inhibitorwhich was Roche Complete Protease Inhibitors (2×) was added to block thetrypsin activity prior to selection on the relevant antigen. Four roundsof selection were performed. Soluble expressed TNFR1 binding dAbs wereassessed using the BIAcore™ for their ability to bind TNFR1 with orwithout the presence of proteases during one hour or overnightincubations at 37° C. in the presence or absence of trypsin (at 100μg/ml or 1000 μg/ml final trypsin concentration).

This led to the isolation of two lead molecules DOM1h-131-202 andDOM1h-131-206 which demonstrated improved protease resistance as shownby BIAcore™ antigen binding experiments. It is interesting to note thatDOM1h-131-202 contained only one mutation in CDR2 (V53D), all amino acidnumbering according to Kabat) in comparison to DOM1h-131-511, whereasDOM1h-131-206 contained only two mutations: the first mutation is thesame as in DOM1h-131-202 (V53D mutation in CDR2) and the second is aY91H mutation in FR3 (see FIG. 3). This Y91H mutation in FR3 does occurin the 3-20 human germline gene indicating that this residue occurs inhuman antibodies. The three clones DOM1h-131-511, DOM1h-131-202 andDOM1h-131-206 have amino acid sequences as shown in FIG. 3.

Activity of the Molecules was Determined as Below:

BIAcore™ binding affinity assessment of DOM1H-131-202, DOM1H-131-511 andDOM1H-131-206 for binding to human TNFR1.

The binding affinities of DOM1H-131-202, DOM1H-131-511 and DOM1H-131-206for binding to human recombinant E. coli-expressed human TNFR1 wereassessed by BIAcore™ analysis. Analysis was carried out usingbiotinylated human TNFR1. 1400 RU of biotinylated TNFR1 was coated to astreptavidin (SA) chip. The surface was regenerated back to baselineusing mild acid elution conditions. DOM1H-131-202, DOM1H-131-511 andDOM1H-131-206 were passed over this surface at defined concentrationsusing a flow rate of 50 μl/min. The work was carried out on a BIAcore™3000 machine and data were analysed and fitted to the 1:1 model ofbinding. The binding data fitted well to the 1:1 model for all testedmolecules. All K_(D) values were calculated from k_(on) and k_(off)rates. BIAcore™ runs were carried out at 25° C.

The data below were produced from three independent experiments. In eachexperiment the results were calculated by averaging a number of fitsusing highest dAb concentrations for kd and lower concentrations for ka.The data are presented as the mean and standard deviation (in brackets)of the results (Table 1).

TABLE 1 BIAcore ™ data for DOM1H-131-202, DOM1H-131-511 andDOM1H-131-206 binding to human TNFR1 k_(on) k_(off) K_(D) (nM)DOM1H-131-511 5.03E+05 5.06E−04 1.07 (511) (1.07E+05) (1.01E−04) (0.44)DOM1H-131-202 1.02E+06 5.42E−04 0.55 (202) (2.69E+05) (3.69E−05) (0.11)DOM1H-131-206 1.55E+06 7.25E−04 0.47 (206) (3.57E+05) (1.95E−04) (0.06)

DOM1H-131-202, DOM1H-131-511 and DOM1H-131-206 bound similarly and withhigh affinity to human TNFR1. DOM1H-131-202 and DOM1H-131-206 bind withaverage affinities of 0.55 nM and 0.47 nM respectively. BothDOM1H-131-202 and DOM1H-131-206 have a slightly better affinity incomparison to DOM1H-131-511 which has an average affinity of 1.07 nM.

Receptor Binding Assay:

The potency of the dAbs was determined against human TNFR1 in a receptorbinding assay. This assay measures the binding of TNF-alpha to TNFR1 andthe ability of soluble dAb to block this interaction. The TNFR1-FCfusion is captured on a bead pre-coated with goat anti-human IgG (H&L).The receptor coated beads are incubated with TNF-alpha (10 ng/ml), dAb,biotin conjugated anti-TNF-alpha and streptavidin alexa fluor 647 in ablack sided clear bottomed 384 well plate. After 6 hours the plate isread on the ABI 8200 Cellular Detection system and bead associatedfluorescence determined. If the dAb blocks TNF-alpha binding to TNFR1the fluorescent intensity will be reduced.

Data was analysed using the ABI 8200 analysis software. Concentrationeffect curves and potency (EC₅₀) values were determined using GraphPadPrism and a sigmoidal dose response curve with variable slope. The assaywas repeated on three separate occasions. A TNF-alpha dose curve wasincluded in each experiment (FIGS. 38 and 39). The concentration ofTNF-alpha used to compete with dAb binding to TNFR1 (10 ng/ml) isapproximately 90% of the maximum TNF-alpha response in this assay.

A representative graph is shown in FIG. 39 showing the ability of dAbsto inhibit the binding of TNF-alpha to TNFR1. In all three experimentsthe negative control samples (HEL4, an anti-hen egg white lysozyme dAband V_(H) dummy) weakly inhibit the interaction between TNF-alpha andTNFR1 at high concentrations. The average potency (EC₅₀) values for thetest samples and positive controls (anti-TNFR1 mAb obtained from R&DSystems, mAb225) and Enbrel™ (etanercept; a dimeric fusion consisting ofTNFR2 linked to the Fc portion of IgG1; licensed for the treatment ofrheumatoid arthritis) are shown in Table 2.

TABLE 2 Potency (EC₅₀) values for DOM1H-131-202, DOM1H-131-206 andDOM1H-131-511 in a TNFR1 receptor binding assay for three repeatexperiments. Sample Average EC₅₀ (nM) SEM DOM1H-131-202 0.11 0.008DOM1H-131-206 0.07 0.01 DOM1H-131-511 0.19 0.01 Enbrel ™ (Etanercept)0.20 0.07 Anti-TNFR1 mAb # mAb225 0.08 0.003

In this assay DOM1H-131-206 appears more potent than the other two dAbsbeing tested and has a similar potency to the commercially availableanti-TNFR1 mAb, MAB225 (R and D Systems).

Expression of Lead Clones from Pichia pastoris was Carried Out asDescribed Below:

The primary amino acid sequence of the three lead molecules was used toproduce codon optimised genes for secreted expression in Pichiapastoris. There is 75% sequence identity between the codon optimized andthe non-codon optimized DOM1H-131-206. The three synthetic genes werecloned into the expression vector pPIC-Zα (from Invitrogen) and thentransformed into two Pichia strains, X33 and KM71H. The transformedcells were plated out onto increasing concentrations of Zeocin (100,300, 600 and 900 μg/ml) to select for clones with multiple integrants.Approximately 15 clones for each cell line and construct were selectedfor expression screening. As the correlation between high/low gene copynumber and expression level is not fully understood in Pichia pastoris,several clones were picked from across the Zeocin concentration range. 5L fermenter runs were carried out using clones that had not beenextensively screened for high productivity. This allowed the productionof significant amounts of material for further studies.

Material Production for Protein Characterisation:

Protein A based chromatography resins have been extensively used topurify V_(H) dAbs from microbial culture supernatants. Although thisallows a single step purification method for producing high puritymaterial, usually >90% in most cases, for some molecules the low pHelution conditions can result in the formation of aggregates. There isalso the issue of the limited capacity of affinity resins for dAbs; thiswould mean the use of significant quantities of resin to process fromfermenters. In order to produce high quality material forcharacterisation and further stability and nebuliser studies, adownstream purification process was devised using a mixed modal chargeinduction resin as the primary capture step followed by anion exchange.Without significant optimisation, this allowed the recovery of ˜70% ofthe expressed dAb at a purity of ˜95%.

For the capture step on the mixed modal charge induction resin, CaptoMMC from GE Healthcare, column equilibration is performed using 50 mMsodium phosphate pH6.0 and the supernatant is loaded without any needfor dilution or pH adjustment. After washing the column, the protein iseluted by pH gradient using an elution buffer which is 50 mM Tris pH9.0. The specific wash and gradient conditions will vary slightlydepending on the pI of the protein being eluted

The eluate peak is then further purified with a flow through step usinganion exchange chromatography. This removes residual HMW contaminationsuch as alcohol oxidase and reduces endotoxin. The resin is equilibratedwith either PBS or phosphate buffer pH 7.4 without salt. Upon loadingthe eluate from Capto MMC onto the anion exchange resin the dAb does notbind and is recovered from the flow through. Endotoxin and othercontaminants bind to the resin. The presence of salt if using PBS bufferimproves protein recovery to 91% for this step rather than 86% recoveryachieved without salt. However the presence of salt reduces theeffectiveness of endotoxin removal such that a typical endotoxin levelof dAb following this step with the inclusion of salt was measured as58EU/ml compared with a level of <1.0EU/ml obtained when no salt waspresent.

Protein Characterisation:

The material produced from the 5 L fermenter runs was characterised foridentity using electrospray mass spectrometry, amino terminal sequencingand isoelectric focusing and for purity using SDS-PAGE, SEC and Gelcodeglycoprotein staining kit (Pierce).

Identity:

The amino terminal sequence analysis of the first five residues of eachprotein, was as expected. Mass spectrometry was performed on samples ofthe proteins which had been buffer exchanged into 50:50 H₂O:acetonitrilecontaining 0.1% glacial acetic acid using C4 Zip-tips (Millipore). Themeasured mass for each of the three proteins was within 0.5Da of thetheoretical mass based on the primary amino acid sequence (calculatedusing average masses) when allowing for a mass difference of −2 from theformation of the internal disulphide bond. IEF was used to identify theproteins based on their pI which was different for each protein.

Purity:

The three proteins were loaded onto non-reducing SDS-PAGE gels in 1 μgand 10 μg amounts in duplicate. A single band was observed in allinstances. Size exclusion chromatography was also performed todemonstrate levels of purity. For size exclusion chromatography (SEC)100 μg of each protein were loaded onto a TOSOH G2000 SWXL columnflowing at 0.5 ml/min. Mobile phase was PBS/10% ethanol.

Investigation of dAb Stability for Candidate Selection:

For the indication of COPD, it would be necessary to deliver the dAbinto the lung, eg using a nebuliser device. This would mean the proteincould potentially experience a range of shear and thermal stressesdepending on the type of nebuliser used and could be subjected toenzymatic degradation by proteases in the lung environment. It wasdetermined if the protein could be delivered using this type of device,form the correct particle size distribution and remain functionalfollowing nebuliser delivery. Therefore the intrinsic stability of eachmolecule to a range of physical stresses was investigated to determinethe baseline stability and the most sensitive stability indicatingassays. As the stability of each protein will be dependent on the buffersolution it is solubilised in, some pre-formulation work was necessary.This information, such as buffer, pH, would also be useful forunderstanding the stability of the protein during the downstreampurification process and subsequent storage. In order to characterisethe changes in the molecules during exposure to a range of physicalstresses, a range of analytical techniques were used such as sizeexclusion chromatography (SEC), SDS-PAGE and isoelectric focusing (IEF).

Assessment of Protease Stability of DOM1H-131-202, DOM1H-131-511 andDOM1H-131-206:

The protease stability of DOM1H-131-202, DOM1H-131-511 and DOM1H-131-206was assessed by BIAcore™ analysis of the residual binding activity afterpre-incubation for defined timepoints in excess of proteases.Approximately 1400RU of biotinylated TNFR1 was coated to a streptavidin(SA) chip. 250 nM of DOM1H-131-202, DOM1H-131-511 and DOM1H-131-206 wasincubated with PBS only or with 100 μg/ml of trypsin, elastase orleucozyme for 1, 3, and 24 hours at 30° C. The reaction was stopped bythe addition of a cocktail of protease inhibitors. The dAb/proteasemixtures were then passed over the TNFR1 coated chip using referencecell subtraction. The chip surface was regenerated with 10 ul 0.1Mglycine pH 2.2 between each injection cycle. The fraction ofDOM1H-131-202, DOM1H-131-511 and DOM1H-131-206 bound to human TNFR1 (at10 secs) pre-incubated with proteases was determined relative to dAbbinding without proteases. BIAcore™ runs were carried out at 25° C.

The data was produced from three independent experiments. The bar graphindicates mean values and the error bars indicate standard deviation ofthe results (for results see FIG. 24).

It was found that DOM1H-131-202 and DOM1H-131-206 were shown to havegreater resistance to proteolytic degradation by trypsin, elastase orleucozyme in comparison to DOM1H-131-511. The difference betweenDOM1H-131-202 and DOM1H-131-206 in comparison to DOM1H-131-511 is mostpronounced after 1 hr with trypsin and after 3 hrs with elastase orleucozyme.

Thermal Stability as Determined Using DSC:

In order to determine at which pH the molecules had the greateststability, differential scanning calorimeter (DSC) was used to measurethe melting temperatures (T_(m)) of each dAb in Britton-Robinson buffer.As Britton-Robinson is made up of three component buffer systems(acetate, phosphate and borate), it is possible to produce a pH rangefrom 3-10 in the same solution. The theoretical pI was determined fromthe proteins primary amino acid sequence. From the DSC, the pH at whichthe dAbs had their greatest intrinsic thermal stability was found to bepH 7 for DOM1H-131-202 (202), pH 7-7.5 for DOM1H-131-206 (206) and pH7.5 for DOM1H-131-511 (511). For all subsequent stress and stabilitywork the following pHs were used for each dAb; for DOM1H-131-202 (202)and DOM1H-131-206 (206) pH 7.0 and for DOM1H-131-511 (511) pH 7.5 inBritton-Robinson buffer. The results are summarised in Table 3 below:

TABLE 3 Summary of the pH and T_(m)s of DOM1H-131-202 (202),DOM1H-131-206 (206) and DOM1H-131-511 (511) as determined by DSC inBritton-Robinson buffer at 1 mg/ml pH that gives greatest Tm (° C.) ofthe dAb intrinsic thermal stability dAb at the given pH DOM1H-131-202(202) 7.0 68.6 DOM1H-131-206 (206) 7.0-7.5 65.8 DOM1H-131-511 (511) 7.558.0Intrinsic Solubility Testing:

All the lead dAbs were concentrated in centrifugal Vivaspinconcentrators (5K cut-off), to determine their maximum solubility andthe levels of recovery upon concentration. Experiments were performed inBritton-Robinson buffer at the most stable pH. Sample volumes andconcentrations were measured over a time course and deviation fromexpected concentration recorded as well as percent recovery of thesample.

It was found that all proteins could be concentrated to over 100 mg/mlin Britton-Robinson buffer. Both DOM1H-131-202 (202) and DOM1H-131-206(206) showed lower recoveries than expected compared to DOM1H-131-511(511), but still within acceptable levels.

Nebuliser Delivery of the Lead dAbs:

By testing different nebulisers and formulation buffers it wasdemonstrated that the dAb could effectively be delivered using a widerange of nebulising devices. More importantly, it was shown for thefirst time that nebulisation of the dAb in the formulation bufferproduced the desired particle size distribution (compared using thepercentage of droplets <5 μm) for effective lung delivery whilstmaintaining protein functionality. This is further described below.

Comparison of Performance in Various Devices:

DOM1H-131-511 (511) was tested in six nebuliser devices comprising twodevices from each of the three main groups of nebulisers for liquidformulations i.e. ultrasonic nebulisers, jet nebulisers and vibratingmesh nebulisers. In each device the dAb was tested at 5 mg/ml with arange of PEG concentrations. For each sample the percentage of dropletsize <5 μm was measured using a Malvern Spraytek Device (MalvernInstruments Limited, UK) and the results are shown in FIG. 35. Thestability of each sample after being nebulised was assessed using SEC toanalyse the amount of sample which had dimerised both in the materialremaining in the cup and in collected aerosol. The results may be seenin FIG. 36. The less the extent of dimer formation the greater thestability.

Most devices can deliver 40% or more of the liquid formulation in thecorrect size range but the eFlow (a vibrating mesh nebuliser device) andPARI LC (a jet nebuliser) devices perform better, with the PARI LC*(star) device delivering more than 80% when PEG is included in thebuffer. This increase in delivery with PEG is also observed with theeFlow and, to a lesser extent, with the PARI LC+.

Importantly activity of the dAb was also found to be retained afternebulisation (see results in FIG. 8)

Effect of Buffer Additives:

Due to the lower stability of DOM1H-131-511 (511), the 50 mM phosphateformulation buffer contained both PEG 1000 and sucrose (and has aviscosity which is within the range which is defined as about equal tothe viscosity of a solution of about 2% to about 10% PEG 1000 in 50 mMphosphate buffer containing 1.2% (w/v sucrose) to help protect the dAbfrom both shear and thermal stress. As both DOM1H-131-202 (202) andDOM1H-131-206 (206) have higher T_(m)'s and showed considerably improvedstability to thermal stress, all the molecules were tested in both theoriginal formulation buffer and in Britton-Robinson buffer (which has alower viscosity than the formulation buffer). The dAbs were tested inboth the E-flow and Pari LC+ devices with run time of 3.5 minutes at aprotein concentration of 5 mg/ml and the particle size distributiondetermined using a Malvern Spraytek Device. As a comparison, a marketeddrug for cystic fiborosis (designated standard protein X) that isdelivered using a nebuliser device, was tested in its own formulationbuffer. The results are shown in FIG. 37. For good delivery anddistribution into the deep lung, the ideal particle size is less than 6microns, e.g. <5 μm. All the dAbs give comparable levels of particlesizes that were less than 5 μm in both the Britton-Robinson buffer andformulation buffer (as described earlier). However, the higher viscosityof the formulation buffer could be particularly beneficial for producingparticles within the correct size range, e.g. particles <5 μm. Theconcentration of the dAb in the cup of the device was determined by A₂₈₀measurements before and after nebulisation. It was found that theprotein concentration did not change significantly indicating thatneither the protein nor vehicle is preferentially nebulised duringdelivery.

Conclusion:

It has been demonstrated as described above that polypeptides such asdAbs can be nebulised in a range of commercially available nebuliserdevices and importantly that they retain stability and biologicalactivity after nebulisation and there is no significant aggregationobserved following nebulisation. When viscosity enhancing excipients,such as PEG are added to the buffer formulation, particle sizedistribution and percentage droplet size less than 5 μm can be improved,thus potentially improving dAb delivery to the deep lung.

Delivery of dAb to the lung can also be improved by increasing the dAbconcentration for example a concentration of up to about 40 mg/ml anddelivery time without any reduction in dAb stability or activity.

Example 1 Phage Vector pDOM13

A filamentous phage (fd) display vector, pDOM13 was used. This vectorproduces fusion proteins with phage coat protein III. The multiplecloning site of pDOM13 is illustrated in FIG. 1. The genes encoding dAbswere cloned as SalI/NotI fragments.

Example 2 Test Protease Selections on Phage-Displayed Domain Antibodies(dAbs) with a Range of Resistance to Trypsin

The genes encoding dAbs DOM4-130-54 which binds IL-1R1, DOM1h-131-511which binds TNFR1, and DOM15-10, DOM15-26 and DOM15-26-501, which bindVEGFA, were cloned in pDOM13 and phages displaying these dAbs wereproduced according to standard techniques. Phages were purified by PEGprecipitation, resuspended in PBS and titered.

The above dAbs displayed a range of ability to resist degradation bytrypsin when tested as isolated proteins. Resistance to degradation wasassessed as follows: dAb (1 mg/ml) in PBS was incubated with trypsin at40 μg/ml at 30° C., resulting in a molecular ratio of 25:1 dAb:trypsin.Samples (30 μl) were taken immediately before addition of trypsin, andthen at T=1 hour, 3 hours, and 24 hours. Protease activity wasneutralized by addition of Roche Complete Protease Inhibitors (2×)followed by immersion in liquid nitrogen and storage on dry ice. 15 μgof each dAb sample was subsequently analyzed by electrophoresis on aNovex 10-20% Tricine gel and proteins were stained with SureBlue (1×).

Both DOM15-10 and DOM15-26-501 were significantly digested during thefirst three hours. DOM15-26, DOM4-130-54 and DOM1h-131-511 were morestable, with digestion of the dAbs only becoming apparent after 24 hours(FIG. 2).

The phage-displayed dAbs were also incubated in the presence of trypsinto evaluate if trypsin resistance of phage-displayed dAbs correlatedwith the results obtained with the isolated soluble dAbs. Variousconcentrations of trypsin and incubation times were tested. In allcases, after neutralization of trypsin with Roche Complete ProteaseInhibitors, the phages were tested for their ability to bind a genericligand: protein A, which binds all V_(H) domain antibodies (e.g.,DOM1h-131, DOM15-26, DOM15-26-501) or protein L, which binds all V_(K)domain antibodies (e.g., DOM4-130-54, DOM15-10). Phage were also testedfor binding to target antigens. In both cases, binding was assumed tocorrelate with retention of the dAb structural integrity throughresistance to proteolysis. The binding activity was measured either byELISA (using conjugated antibodies against phage) or by elution of boundphages and titre analysis following infection of exponentially growingE. coli TG1 cells.

Tests with DOM15-10, DOM15-26 and DOM15-26-501 on Phage

Each dAb was treated for one hour at room temperature with a range oftrypsin concentrations (100 μg/ml, 10 μg/ml and 0 μg/ml). Trypsinactivity was blocked with Roche Complete Protease Inhibitor (1×) andthen the phages were diluted in 2% Marvell in PBS, incubated with 50 nMof biotinylated antigen (recombinant human VEGF (R&D systems)) for onehour at room temperature. Strepavidin-coated beads (Dynabeads M-280(Invitrogen)) that were pre-blocked for one hour at room temperaturewith 2% Marvell in PBS were added, and the mixture was then incubatedfor five minutes at room temperature. All of the incubation steps withDynabeads were carried out on a rotating wheel. Unbound phages werewashed away by washing the beads eight times with 1 ml of 0.1% Tween-20in PBS. Bound phages were eluted with 0.5 ml of 0.1M Glycine pH2.2 andneutralized with 100 μl of 1M Tris-HCL pH 8.0. Eluted phage were used toinfect exponentially growing TG1 cells (one hour at 37° C.) and platedon Tetracycline plates. Plates were incubated overnight at 37° C. andcolony counts were made (see Table 4). The best results were observedfrom selection with incubation with 100 μg/mltrypsin. There was about a10-fold increase in the yield of DOM15-26 in comparison to DOM15-10 andDOM15-26-501.

A second experiment was done to further confirm these results under moresevere incubation conditions. Phage displayed dAbs were treated for 1hour or 2 hours at 37° C. with agitation (250 rpm). The best resultswere observed from selections with 2 hour incubation with 100ug/mltrypsin. The yield of DOM15-26 was 200-fold higher than the yieldof DOM15-26-501 and 1000-fold higher than the yield of DOM15-10.

In a third experiment, phages displaying DOM15-26 and DOM15-26-501 weremixed 1:1 at the start. They were then either incubated with trypsin(1000 μg/ml) or without trypsin for two hours at 37° C. with agitation(250 rpm), and then selected for antigen binding as described above.Sequencing of ten colonies from each selection revealed a mixedpopulation of clones for selection without trypsin pre-treatment(DOM15-26: 4/10; DOM15-26-501: 6/10), whereas all clones from theselection with trypsin encoded for DOM15-26 as expected.

These experiments indicate that a selection pressure can be obtained byadding a protease to phages displaying dAbs, such that phages displayingthe most proteolytically stable dAbs are preferentially selected(following panning on a generic ligand or the antigen).

TABLE 4 Length of Trypsin DOM15-26 DOM15-26-501 1:1 mixed DOM15-10Experiment incubation Temp. concentration titre titre titre titre 1 1 hrRoom temp 100 μg/ml 1.6 × 10⁸ 6.3 × 10⁷ 1.1 × 10⁷ input 10¹⁰ 1 hr Roomtemp 10 μg/ml   3 × 10⁸ 4.4 × 10⁸ 2.4 × 10⁸ 1 hr Room temp 0 μg/ml 0.9 ×10⁸   2 × 10⁸ 0.7 × 10⁸ 2 1 hr, 250 rpm 37° C. 100 μg/ml   2 × 10⁷   1 ×10⁶   1 × 10⁵ input 10⁹ 2 hr, 250 rpm 37° C. 100 μg/ml   1 × 10⁷   6 ×10⁴   1 × 10⁴ 2 hr, 250 rpm 37° C. 0 μg/ml 5.4 × 10⁷ 4.1 × 10⁷   3 × 10⁸3   2 h, 250 rpm 37° C. 100 μg/ml 2.3 × 10⁸   8 × 10⁵ 6.8 × 10⁷ input10¹⁰   2 h, 250 rpm 37° C. 0 μg/ml 3.9 × 10⁸ 4.4 × 10⁸ 4.8 × 10⁸

Tests with DOM4-130-54 on Phage

DOM4-130-54 was tested in a similar protocol as described above. Theparameters that were varied were: concentration of trypsin, temperatureand length of incubation. Biopanning was done against IL-RI-Fc (a fusionof IL-1RI and Fc) at 1 nM concentration in PBS. Significant reductionsin phage titre were only observed after incubation of the phage with 100μg/ml trypsin overnight at 37° C. (see Table 5).

TABLE 5 Length of incubation Temperature Trypsin concentration Titre 1hr Room temp 100 μg/ml  1.8 × 10¹⁰ 1 hr Room temp  10 μg/ml  7.2 × 10⁹ 1hr Room temp  0 μg/ml  6.6 × 10⁹ Overnight Room temp 100 μg/ml 2.16 ×10⁹ Overnight Room temp  10 μg/ml  7.2 × 10⁹ Overnight Room temp  0μg/ml  7.8 × 10⁹ Overnight 37° C. 100 μg/ml 2.04 × 10⁶ Overnight 37° C. 10 μg/ml 3.84 × 10⁸ Overnight 37° C.  0 μg/ml  7.2 × 10⁹

Tests with DOM1h-131 Phage

DOM1h-131 phage (closely related to DOM1h-131-511 by amino acidsequence) were treated with 0 μg/ml, 10 μg/ml, 100 μg/ml and 1000μg/mltrypsin for one hour at room temperature. Digestion was inhibitedby the addition of 25× Complete Protease Inhibitors (Roche). Serial2-fold dilutions of the phage were carried out down an ELISA platecoated with 1 nM TNFR1, and binding phage were detected withanti-M13-HRP. The results are shown below in Table 6.

TABLE 6 DOM1h-131 Trypsin concentration 1 100 10 0 Phage mg/ml μg/mlμg/ml μg/ml input 0.284 0.418 0.784 0.916 4.51E+10 0.229 0.377 0.8020.944 2.26E+10 0.183 0.284 0.860 0.949 1.13E+10 0.133 0.196 0.695 0.9625.64E+09 0.114 0.141 0.573 0.946 2.82E+09 0.089 0.115 0.409 0.8501.41E+09 0.084 0.084 0.286 0.705 7.05E+08 0.080 0.084 0.213 0.5773.52E+08

These test experiments clearly show that 100 μg/ml of trypsin and atemperature of 37° C. are appropriate to apply a selection pressure onphages displaying dAbs of various degrees of resistance to proteolysisby trypsin. Incubation time with the protease can be optimized for eachphage-displayed dAb, if desired.

Example 3 Protease Selection of Phage-Displayed Repertoires of DomainAntibodies

Four repertoires were created using the following dAbs as parentmolecules: DOM4-130-54, DOM1h-131-511, DOM15-10 and DOM15-26-555. Randommutations were introduced in the genes by PCR using the StratageneMutazyme II kit, biotinylated primers and 5-50 pg of template for a 50μl reaction. After digestion with SalI and NotI, the inserts werepurified from undigested products with streptavidin-coated beads andligated into pDOM13 at the corresponding sites. E. coli TB1 cells weretransformed with the purified ligation mix resulting in largerepertoires of tetracycline-resistant clones: 8.5×10⁸ (DOM4-130-54),1.5×10⁹ (DOM1h-131-511), 6×10⁸ (DOM15-10) and 3×10⁹ (DOM15-26-555).

Phage libraries were prepared by double precipitation with PEG andresuspended in PBS.

The rates of amino acid mutations were 2.3 and 4.4 for the DOM1h-131-511and DOM4-130-54 repertoires, respectively. The functionality wasassessed by testing 96 clones in phage ELISA using wells coated withprotein A or protein L (at 1 μg/ml). 62.5% and 27% of the clonesexhibited functional display of dAbs in the DOM1h-131-511 andDOM4-130-54 repertoires, respectively.

The rates of amino acid mutations were 2.5 and 4.6 for the DOM15-10 andDOM15-26-555 repertoires, respectively. The functionality was assessedby testing 96 clones in phage ELISA using wells coated with protein A orprotein L (at 1 μg/ml). 31.3% and 10.4% of the clones exhibitedfunctional display of dAbs in the DOM15-10 and DOM15-26-555 repertoires,respectively.

DOM4-130-54 and DOM1h-131-511 Repertoires

Four rounds of selection were carried out with these libraries to selectfor dAbs with improved protease resistance.

The first round of selection was by antigen binding (1 nM or 10 nMantigen) without protease treatment to clean-up the library to removeany clones that no longer bound antigen with high affinity. The outputsfrom round 1 were in the 10⁸-10¹⁰ range (compared to an input of 10¹¹phage) indicating that the majority of the library bound antigen withhigh affinity.

In round 2, protease treatment with 100 μg/mltrypsin was introduced, andthe outputs are as shown below in Table 7:

TABLE 7 Trypsin incubation DOM1h-131-511 DOM4-130-54 conditions librarylibrary 37° C. overnight 1.86 × 10⁶  2.1 × 10⁶ 37° C. 2 hrs 4.8 × 10⁸5.1 × 10⁸ Room temperature 2 hrs 1.2 × 10⁹ 4.62 × 10⁹  No trypsin  ~1 ×10⁹  ~4 × 10⁹ No antigen 1.8 × 10⁴  <6 × 10³

There was significant selection when the dAbs were treated with trypsinat 37° C. overnight. This output was taken forward to round 3, where thephage were treated with either 1 mg/ml or 100 μg/ml trypsin at 37° C.for 24 hours. The titres of the trypsin treated phage from round 3 were10⁵-10⁶ for the DOM1h-131-511 repertoire and 10⁷-10⁸ for theDOM4-130-154 repertoire.

All outputs from round 3 (DOM1h-131-511 and DOM4-130-154 with 1 mg/mland 100 μg/ml) underwent a fourth round of selection against 1 nMantigen with 100 μg/ml trypsin. The titres were in the range of 10⁶-10⁸,similar to that seen in round 3. Some enrichment was seen for theDOM1h-131-511 repertoire, but no enrichment was seen for the DOM4-130-54repertoire.

DOM15-10 and DOM15-26-555 Repertoires

The first round of selection was carried out with 2 nM biotinylatedhVEGF (human vascular endothelial growth factor) concentration andwithout protease treatment to clean-up the library to remove any clonesthat no longer bound antigen with high affinity. The outputs from round1 were about 10⁸ (compared to an input of 10¹⁰ phage for DOM15-10 and10¹¹ phage for DOM15-26-555) indicating that the majority of the librarybound antigen with high affinity.

The second and third rounds of selection were performed with 2 nMbiotinylated hVEGF. Prior to panning on hVEGF, the phages were incubatedin the presence of trypsin (100 μg/ml) at 37° C. in a shaker (250 rpm).Incubation time was one hour for the DOM15-10 repertoire and two hoursfor the DOM15-26-555 repertoire.

The outputs were as follows: 1.5×10⁶ and 9×10⁵ for the second and thirdrounds of selection with the DOM15-10 repertoire; 2.2×10⁸ and 3.9×10⁹for the second and third rounds of selection with the DOM15-26-555.

Example 4

Analysis of selection outputs: DOM4-130-54 and DOM1h-131-511 Repertoires

All outputs from round 3 and round 4 were subcloned into the pDOM5vector and transformed into JM83 cells. The pDOM5 vector is apUC119-based vector. Expression of proteins is driven by the Placpromoter. A GAS1 leader sequence (see WO 2005/093074) ensured secretionof isolated, soluble dAbs into the periplasm and culture supernatant ofE. coli JM83. 96 and 72 individual colonies from round 3 and round 4were randomly picked for expression

12-24 clones were sequenced from each round 3 and round 4 output.Consensus mutations were observed in both selections and approximately25 clones harboring consensus motifs were chosen for furthercharacterization. The amino acid sequences of these clones are shown inFIG. 3 (DOM1h-131-511 selected variants) and FIG. 4 (DOM4-130-54selected variants) and listed as DNA sequences in FIGS. 19A-19L. Theamino acids that differ from the parent sequence in selected clones arehighlighted (those that are identical are marked by dots). The loopscorresponding to CDR1, CDR2 and CDR3 are outlined with boxes.

These clones were expressed in a larger amount, purified on protein L(for DOM4-130-54 variants) and protein A (for DOM1h-131-511 variants)and tested for antigen binding on BIAcore after one hour or overnightincubation at 37° C. in the presence or absence of trypsin (100 μg/ml or1000 μg/ml final concentration).

Generally, the outputs from the DOM4-130-54 selections were more stablewith most clones remaining resistant to trypsin for one hour and thebest clones resistant overnight. In comparison, a small number of clonesfrom the DOM1h-131-511 selections were resistant to trypsin for onehour, whilst none of the clones were resistant overnight.

Example 5 Analysis of Selection Outputs: DOM15-10 and DOM15-26-555Repertoires

The effectiveness of selection with trypsin pre-treatment was firsttested on monoclonal phage ELISA with and without trypsin digestion.Eighteen colonies from the second round of selection and 24 coloniesfrom the third round of selection of each library were picked. ClonesDOM15-10, DOM15-26-501 and DOM15-26 were used as controls. Additionalcontrols included amplified and purified phage solution from eachlibrary after second and third rounds of trypsin selection.

Each phage sample was divided into two fractions, the first was treatedwith 100 ug/mltrypsin, the second was not treated with trypsin.Incubation of both fractions was carried out for one hour at 37° C. withagitation (250 rpm) and blocked by adding Roche Complete ProteaseInhibitor (1×).

Phage ELISA was performed using the trypsin-digested and undigestedsamples. ELISA wells were coated with neutravidin in 0.1M bicarbonatebuffer at a concentration of 1 μg/ml. After the washing steps with PBSand blocking of the antigen-coated wells with 1% Tween-20 in PBS for onehour at room temperature, the wells were coated with biotinylated hVEGFdiluted in 1% Tween-20 in PBS at a concentration of 100 ng/ml. Next, thewells were washed with PBS and treated or untreated phage supernatantsdiluted 1:1 with 1% Tween-20/PBS, were added. After 30 minutes ofincubation at 37° C., the wells were washed with 1% Tween-20/PBS,followed by a 30 minute incubation at 37° C. with anti-M13 phage-HRPconjugate (diluted 1/5000 in 1% Tween-20/PBS). The wells were thenwashed with PBS and peroxidase. Reaction was initiated by addingSureBlue reagent. After about ten minutes, the reaction was stopped withan equivalent volume of 1M HCl and the wells were read at OD_(450nM).

ELISA read-outs of unstable controls DOM15-10 and DOM15-26-501 treatedwith trypsin gave an OD₄₅₀ lower than 0.404 and this value was assumedas a border value of an unstable clone. All samples that gave an ODlower than 0.404 were considered to be unstable. All samples above thatvalue were considered to be stable.

TABLE 8 Trypsin No trypsin 2nd 3rd 2nd 3rd Library selection selectionselection selection DOM15-10   33% 89% 100% 100% DOM15-26-555 94.4% 100%100% 100%

Table 8 shows the percentage of stable clones after the second and thirdrounds of trypsin selection of each library. The enrichment of trypsinresistant clones is visible in both libraries after the third round ofselection. The values of control ELISA wells containing amplifiedpurified phage mix after each selection were much higher than 0.404 ineach case after trypsin digestion. Moreover, a small increase in signalwas observed when comparing trypsin-treated phage from the third roundof selection with trypsin-treated phage from the second round ofselection. The DOM15-10 phage library showed an increase of about 14% ofthe starting value. DOM15-26-555 phage library showed an increase thatrepresents about 2% of the starting value.

Overall these results show that selection with trypsin pre-treatment waseffective to select trypsin-resistant phage clones from the DOM15-10 andDOM15-26-555 repertoires.

All outputs from the second and third rounds of selection (DOM15-26-555)and from the third round of selection only (DOM15-10) were subclonedinto the pDOM5 vector and transformed into HB2151 electrocompetentcells. The pDOM5 vector is a pUC119-based vector. Expression of proteinsis driven by the Plac promoter. A GAS1 leader sequence ensured secretionof isolated, soluble dAbs into the periplasm and culture supernatant ofE. coli HB2151. 184 individual colonies from each round of selection (3and 4) were randomly picked for expression in 1 ml culture volumes.

Bacterial supernatants were diluted in HBS-EP BIAcore buffer (1:1 volumeratio) and split to duplicates. Trypsin was added to only one vial at afinal concentration of 20 μg/ml. Incubation was carried out for 40minutes at 37° C. with agitation (250 rpm). After blocking the reactionwith Roche Complete Protease Inhibitor (1×), both trypsin treated anduntreated phage supernatants were tested on BIAcore 3000 for antigenbinding (2,000 RU of biotinylated hVEGF on a SA sensorchip).

The criteria for picking clones were: a decrease in antigen binding of<15% of dAbs treated with trypsin relative to untreated dAbs (based onmax RU reached on selected time point), which would reflect dAbsstability to protease treatment in general; and off-rate decrease of<40% between two time points during dissociation of a dAb from theantigen. Based on these values, 60 clones from both the second and thirdrounds of selection of the DOM15-26-555 library and 17 clones from thethird round of selection of the DOM15-10 library were sequenced.Consensus mutations were observed in both libraries' outputs and 17clones from each library harboring consensus motifs were chosen forfurther characterization. The amino acid sequences of these clones areshown in FIG. 5 (DOM15-26-555 selected variants) and FIG. 6 (DOM15-10selected variants) and listed as DNA sequences in FIGS. 20A-20E. Theamino acids that differ from the parent sequence in selected clones arehighlighted (those that are identical are marked by dots). The loopscorresponding to CDR1, CDR2 and CDR3 are outlined by boxes.

These clones were expressed in 50 ml expression cultures, purified onprotein A (for DOM15-26-555 variants) or protein L (for DOM15-10variants) diluted to 100 nM concentration in HBS-EP buffer and testedfor antigen binding on BIAcore after 1.5 hours of incubation at 37° C.with agitation (250 rpm) in the presence or absence of trypsin (20 μg/mlfinal concentration).

These clones were also tested for trypsin resistance using the methoddescribed in Example 2. Proteins were buffer exchanged to PBS andconcentrated to 1 mg/ml. 25 μg of protein was mixed with 1 μg of trypsin(Promega) and incubated for 0 hours and 24 hours at 30° C. After thistime, the reaction was blocked with Roche Complete Protease Inhibitor(1×) and DTT, as well as loading agent, was added Samples were denaturedfor five minutes at 100° C. Then 15 μg of each sample was analyzed byelectrophoresis on Novex 10-20% Tricine gels and proteins were stainedwith SureBlue (1×).

Generally, the outputs from the DOM15-26-555 selections were morestable, with most clones remaining resistant to trypsin for 1.5 hourswhen tested on BIAcore and overnight when run on SDS-PAGE. Incomparison, only a small number of clones from the DOM15-10 selectionswere resistant to trypsin for overnight treatment when run on SDS-PAGE.

Example 6 Identification of DOM1h-131-511 Variants

DOM1h-131-203, DOM1h-131-204 and DOM1h-131-206 were analyzed in furtherdetail. They were compared on the BIAcore at a dAb concentration of 500nM after incubation with different concentrations of trypsin (rangingfrom 0 to 100 μg/ml) overnight at 37° C. The BIAcore traces are shown inFIG. 7. The results clearly show that both variants are more resistantthan their parent to proteolysis at high concentration of trypsin (100μg/ml). Two of the dAbs, DOM1h-131-202 and DOM1h-131-206, were alsocompared along with their parent against a range of other proteasesincluding leucozyme, elastase and pancreatin under the conditionsdescribed above, with a protease concentration of 100 μg/ml. The dAbsshowed increased resistance to proteolysis compared to the parentagainst all proteases tested. The BIAcore traces for elastase andleucozyme are shown in FIG. 8.

5 μM of each dAb was treated with 100 μg/ml sequencing grade trypsin for0, 1, 3 and 24 hours. The reaction was inhibited with 25× Roche CompleteProtease Inhibitor and the reactions were run on a 4-12% Novex Bis-Trisgel. The gels are shown in FIG. 9.

Example 7 Identification of DOM4-130-54 Variants

DOM4-130-201 and DOM4-130-202 were analyzed in further detail. They werecompared on the BIAcore at a dAb concentration of 500 nM afterincubation with different concentrations of trypsin (ranging from 0 to100 μg/ml) overnight at 37° C. The BIAcore traces are shown in FIG. 10.The results clearly show that all three variants are more resistant thantheir parent to proteolysis at high concentrations of trypsin (100μg/ml). DOM4-130-201 and DOM4-130-202 were also compared with the parentagainst a range of other proteases including leucozyme, elastase andpancreatin under the conditions described above with a proteaseconcentration of 100 μg/ml. Although the results were less apparent thanwith trypsin, the lead dAbs showed increased resistance to proteolysiscompared to parent against all proteases tested. The BIAcore traces forelastase and leucozyme are shown in FIG. 11.

5 μM of each dAb was treated with 100 ug/ml sequencing grade trypsin for0, 1, 3 and 24 hours. The reaction was inhibited with 25× Roche CompleteProtease Inhibitor and the reactions were run on a 4-12% Novex Bis-Trisgel. The gels are shown in FIG. 9.

Example 8 Further Characterization of DOM1h-131-511 and DOM4-130-54Variants

The dAbs were first analyzed using Differential Scanning calorimetry(DSC) to determine whether the increase in trypsin resistance correlatedwith an increase in melting temperature (Tm). An increase in trypsinstability does correlate with an increase in Tm (see Table 9)

TABLE 9 Name Tm, ° C. DOM1h-131-511 57.9 DOM1h-131-202 67.5DOM1h-131-203 65.7 DOM1h-131-204 62.3 DOM1h-131-206 64.9 DOM4-130-5454.1 DOM4-130-201 64.7 DOM4-130-202 64.5

The DOM1h-131-511 derived dAbs were also compared in a MRC-5 cell-basedassay (see Table 10). In this assay, the ability of the dAbs toneutralize TNFα stimulated IL-8 release was measured to determinewhether the increase in trypsin stability had led to a decrease inefficacy. However, the activity of the trypsin-resistant dAbs in theassay was substantially unaffected.

TABLE 10 Sample ND50 nM DOM1h-131-511 1.98 DOM1h-131-511 1.71DOM1h-131-511 (230307CE) 1.89 DOM1h-131-203 (230307CE) 2.28DOM1h-131-204 (230307CE) 1.89 DOM1h-131-511 1.46 DOM1h-131-206(230307CE) 0.71

The DOM4-130-54 derived dAbs were tested in a Receptor Binding Assay tosee if they still had the same ability to inhibit the binding of IL-1 toIL-RI (see Table 11). The activity of the trypsin resistant dAbs wasunaffected in this assay.

TABLE 11 dAb IC50 (nM) DOM4-130-54 280 pM DOM4-130-201 257 pMDOM4-130-202 254 pM

Example 9 Identification of DOM15-26-555 Variants

DOM15-26-588, DOM15-26-589, DOM15-26-591, and DOM15-26-593 were analyzedin further detail together with their parent and two additional dAbs,DOM15-26-594 and DOM15-26-595, which were created by mutagenesis tocombine mutations that would have the greatest impact on potency andstability (E6V and F100S/I). Sequences are shown in FIG. 12. Clones werecompared on the BIAcore for hVEGF binding at the dAb concentration of100 nM after incubation with trypsin at a concentration of 200 μg/ml.The reaction was carried out for three hours and 24 hours at 37° C. withagitation (250 rpm). The BIAcore traces of the best clone, DOM15-26-593,and the parent are shown in FIG. 13. Other results are presented as achart in FIG. 14. The results clearly show that all variants are moreresistant than the parent to proteolysis after 24 hours of trypsintreatment.

Trypsin resistance of DOM15-26-593 and the parent was also examined byrunning treated and un-treated samples on SDS-PAGE. Briefly, proteinswere buffer exchanged to PBS and concentrated to 1 mg/ml. 25 ug ofprotein was mixed with 1 μg of sequencing grade trypsin (Promega) andincubated for 0 hours, 1 hour, 3 hours and 24 hours at 30° C. After thistime, the reaction was blocked with Roche Complete Protease Inhibitor(1×) and DTT, as well as loading agent, was added. Samples weredenatured for five minutes at 100° C. 15 ug of each sample was loaded onNovex 10-20% Tricine gels and proteins were stained with SureBlue (1×).The results are shown in FIG. 15. The trypsin resistance profile ofDOM15-26-593 in this experiment varied from the profile shown by theBIAcore experiment, suggesting that differences in reaction conditionsmay influence the final result of trypsin cleavage. Nonetheless,DOM15-26-593 has better biophysical properties, as well as affinity,than other selected clones, as shown below. A summary of the propertiesof the DOM15-26-555 variants is also shown in the table 12 below.

TABLE 12 Attribute SEC-MALLS DSC BIAcore Trypsin Stability dAb % monomerEst. mw Tm ° C. RBA nM KD nM % binding @ + 24 hrs 15-26 0 37-136 64 1028.2 30 15-26-501 0-40 18-290 51 1.14 9.1 5 15-26-555 0 28-78  63 11.726.1 10 15-26-588 10 33 70 27 59.1 15 15-26-589 90 17 63 1.94 9.6 6515-26-591 20 21-234 63 16 38 35 15-26-593 80 17 65 0.323 3.2 8015-26-595 60 17 65 0.828 5 70

Example 10 Identification of DOM15-10 Variants

DOM15-10-11 was analyzed in further detail, together with its parent,DOM15-10. Sequences are shown in FIG. 16. The dAbs were compared on theBIAcore for hVEGF binding at the dAb concentration of 100 nM afterincubation with trypsin at a concentration of 200 μg/ml. The reactionwas carried out for 1 hour, 3 hours and 24 hours at 37° C. withagitation (250 rpm). The BIAcore traces of these dAbs are shown in FIG.17. The result clearly shows that the selected variant is more resistantthan the parent to proteolysis after 24 hours of trypsin treatment.

Trypsin resistance of the lead and the parent was also examined byrunning treated and un-treated samples of SDS-PAGE. Briefly, proteinswere buffer exchanged to PBS and concentrated to 1 mg/ml. 25 μg ofprotein was mixed with 1 μg of sequencing grade trypsin (Promega) andincubated for 0 hours, 1 hour, 3 hours and 24 hours at 30° C. After thistime, the reaction was blocked with Roche Complete Protease Inhibitor(1×) and DTT, as well as loading agent, was added. Samples weredenatured for five minutes at 100° C. 15 μg of each sample was loaded onNovex 10-20% Tricene gels and proteins were stained with SureBlue (1×).The results are presented in FIG. 18. In this case, the trypsinresistant profile correlates well with the BIAcore trypsin test, showingthat the binding activity directly reflects the protein's integrity.

Example 11 Further Characterization of DOM15-26-555 and DOM15-10Variants

The dAbs were analyzed using Differential Scanning calorimetry (DSC) todetermine whether the increase in trypsin resistance correlated with anincrease in Tm. The results are shown in Table 13. There is acorrelation between the trypsin resistance of DOM15-26-555 variants andmelting temperature. The lead DOM15-26-588 and DOM15-26-593 showedimproved Tm, but the other clones did not. It is worth noting that bothDOM15-26-555 and DOM15-10 parent molecules have much higher Tm at thestart (63.3-63.7° C.) than the DOM4-130-54 and DOM1h-131-511 parentmolecules (Tm at start: 57.9-54.1° C.), but overall the proteaseresistant clones reach a Tm in a similar range (average Tm of 65.1° C.for the DOM1h-131-511/DOM4-130-54 variants and average Tm of 64.9° C.for the DOM15-26-55/DOM15-10 variants).

TABLE 13 Name Tm ° C. DOM15-26-555 63.3 DOM15-26-588 70.1 DOM15-26-58963 DOM15-26-591 63 DOM15-26-593 65 DOM15-10 63.7 DOM15-10-11 63.3

The dAbs were also compared in a receptor binding assay and BIAcorekinetics were measured to determine whether the increase in trypsinstability had led to a decrease in efficacy. However, the activity ofthe dAbs in the assay was substantially unaffected or even improved. Theresults are presented in Table 14.

TABLE 14 Clone ID EC₅₀ (nM) K_(D) (nM) DOM15-26-555 11.7 26.1DOM15-26-588 27 59.1 DOM15-26-589 1.94 9.6 DOM15-26-591 16 38DOM15-26-593 0.323 3.2 DOM15-26-594 4.09 15.1 DOM15-26-595 0.828 5DOM15-10 10.23 23.6 DOM15-10-11 3.58 14.6Advantages of an Enhanced Tm

Most proteins—including domain antibodies—exist in two states: a foldedstate

(which leads to a biologically active molecule) and an unfolded state(which does not bear functional activity). These two states co-exist atall temperatures and the relative proportion of each state is usuallydetermined by a constant K that is a function of the kinetic constantsof folding and unfolding. The melting temperature is usually defined asthe temperature at which K=1, i.e. the temperature at which the fractionof folded protein is equal to be fraction of unfolded protein. Theconstant K is determined by the stabilizing and destabilizingintramolecular interactions of a protein and therefore is primarilydetermined by the amino acid sequence of the protein. Extrinsicparameters such as temperature, pH, buffer composition, pressureinfluence K and therefore the melting temperature.

Unfolded proteins are easy targets for degradation mechanisms: (i)exposure of disulfide bonds increase risks of oxidation or reductiondepending on the circumstances, (ii) enhanced backbone flexibilityfavours auto-proteolytic reactions, (iii) exposure of peptide segmentsoffers targets to proteases in vivo, to proteases during productionprocesses and to carry-over proteases during downstream processing andlong-term storage, and (iv) exposure of aggregation-prone segments leadsto inter-molecular aggregation and protein precipitation. In all cases,a loss of protein integrity, protein content and protein activityhappens, thereby compromising efforts to (i) ensure batchreproducibility, (ii) ensure long-term stability on shelf, and (iii) invivo efficacy.

In nature proteins have been designed by evolution to adequately performat body temperature and to be readily replaced via homeostaticmechanisms. Therapeutic proteins manufactured through biotechnogicalprocesses face a different environment: they are frequently produced byrecombinant DNA technology in a foreign host, are expressed at higheramount in large vessels, undergo very important changes in pH or buffercomposition throughout downstream processes and finally are stored athigh concentrations in non-physiological buffers for prolonged period oftime. New delivery techniques (e.g. inhalation, sc patch, slow deliverynanoparticles) are also adding on the stress undergone by therapeuticproteins. Finally the advent of protein engineering techniques hasresulted in the production of enhanced or totally novel therapeuticproteins. Because most engineering techniques are in-vitro basedtechniques aimed at altering or creating new amino acid sequences,evolution processes that have gradually improved biological proteins donot take place, hence resulting in proteins of sub-optimal performanceswith regards to stress resistance.

The technique of the present invention aims at reproducing one of theconditions faced by proteins throughout Darwinian evolution. Peptides orpolypeptides, eg immunoglobulin single variable domains are infused withproteases that play a major role in tissue remodelling and proteinhomeostasis. Any particular mutation that may result in a protein withan improved fit to its function is also tested for its ability to fitwithin the environment it is performing in. This process is reproducedin one embodiment of the present invention: a repertoire of peptide orpolypeptide variants is created and exposed to a protease. In a secondstep, the repertoire of variants is contacted with a specific target.Only those protein variants that have sustained degradation by theprotease are able to engage with the target and therefore recovered, eg,by a simple affinity purification process named ‘biopanning’. The systemoffers a number of advantages in comparison to in vivo processes: theprotein repertoire can be faced with a wider range of conditions, eg arange of proteases, at higher concentrations, for longer times, indifferent buffers or pHs and at different temperatures. Thus this invitro technology offers a means to design proteins that may perform andremain stable in a wider range of environments than those they originatefrom. Clearly this offers multiple advantages for the biotechnologicalindustry and for the area of therapeutic proteins in particular.

Example 12 PK Correlation Data for Protease Resistant Leads

The parent dAb and a protease-resistant dAb in each of the four dAblineages, were further evaluated in vivo (see Table 15 below for listand details)

TABLE 15 Resistance Tm Activity ID as Fc Lineage dAb ID to trypsin (°C.) (nM) fusion DOM4-130 DOM4-130-54 Good 54 0.128* DMS1541 DOM4-130-202Very high 64 0.160* DMS1542 DOM1h-131 DOM1h-131-511 Good 57 0.048†DMS1543 DOM1h-131-206 Very high 64 0.047† DMS1544 DOM15-10 DOM15-10 Low64 0.913† DMS1546 DOM15-10-11 High 63 0.577† DMS1531 DOM15-26DOM15-26-501(*) Low 52 0.330† DMS1545 DOM15-26-593 High 65 0.033†DMS1529 (*)as determined by MRC5/IL-a bioassay; †as determined by RBAassay Note: DOM15-26-501 is a parent version of DOM15-26-555 exemplifiedabove in this patent application. DOM15-26-555 has one germline aminoacid mutation in CDR1 (I34M). DOM15-26-501 has a lower meltingtemperature than DOM15-26-555 (52C v 63.3C) and an increasedsusceptibility to digestion by trypsin. DOM15-26- 501 was chosen overDOM15-26-555 for the PK study as it is a better representative for poorstability in comparison to DOM15-26-593.

We can translate the resistance as follows:

1 is low

2 is moderate

3 is good

4 is high

5 is very high

Then this means that the trypsin resistance of the parent molecules is:

DOM4-130-54 is Good DOM1h-131-511 is Good DOM15-10 is Low DOM15-26-501is Low

As for the selected leads:

DOM4-130-202 is Very high DOM1h-131-206 is Very high DOM15-10-11 is HighDOM15-26-593 is High

Because domain antibodies are small in size (12-15 kDa) they are rapidlycleared from the circulation upon iv or sc injection. Indeed the renalglomerular filtration cut-off is above 50 kDa and therefore smallproteins such as dAbs are not retained in the circulation as they passthrough the kidneys. Therefore, in order to evaluate the long termeffects of resistance to proteases in vivo, we tag domain antibodieswith a moiety that increases systemic residence. Several approaches(e.g. PEG, Fc fusions, albumin fusion, etc) aiming at extendinghalf-life have been reported in the literature. In this application thedomain antibodies have been tagged (or formatted) with the Fc portion ofthe human IgG1 antibody. This format offers two advantages: (i) themolecular size of the resulting dAb-Fc is ˜75 kDa which is large enoughto ensure retention in circulation, (ii) the antibody Fc moiety binds tothe FcRn receptor (also know as “Brambell” receptor). This receptor islocalized in epithelial cells, endothelial cells and hepatocytes and isinvolved in prolonging the life-span of antibodies and albumin: indeedupon pinocytosis of antibodies and other serum proteins, the proteinsare directed to the acidified endosome where the FcRn receptorintercepts antibodies (through binding to the Fc portion) before transitto the endosome and return these to the circulation. Thus by tagging theFc portion to the dAb, it is ensured that the dAbs will exposed for longperiod to two at least compartments—the serum and the pre-endosomalcompartments, each of which containing a specific set of proteolyticenzymes. In addition, the FcRn receptor mediates transcytosis wherebyFc-bearing proteins migrate to and from the extravascular space.

Formatting with Fc was accomplished by fusing the gene encoding the VHand VK dAbs to the gene encoding the human IgG1 Fc, through a shortintervening peptide linker (in bold):

For a VH dAb (underlined):

EVQ......GQGTLVTVSSASTHTCPPCPAPELLGGP... (hIgGlFc)...PGK*

For a VK dAb (underlined):

DIQ.........GQGTKVEIKRTVAAPSTHTCPPCPAPELLGGP... (hIgGlFc)...PGK*

Material was produced by transient transfection of HEK293/6E cells using293-fectin (Invitrogen) according to standard protocols. These cells aredesigned for high-level transient expression when used in conjunctionwith the pTT series of vectors (Durocher et at 2002). Thus the dAb geneswere cloned into a modified pTT5 vector (pDOM38) to generate the Fcfusion expression vector (see FIG. 21). The supernatant from thetransfected cells was harvested at 5 days post-transfection, clarifiedby centrifugation and filtered through a 0.2 μm filter. The dAb-Fcfusion proteins were purified by capture onto Protein-A streamline resin(GE Healthcare). Protein was eluted from the column in 10 mM sodiumcitrate pH3, followed by the addition of and 1M sodium citrate pH6, toachieve a final composition of 100 mM sodium citrate pH6.

The dAb-Fc molecules were tested for in vivo half life in the rat at atarget dose of 5 mg/kg into female Sprague-Dawley rats (n=3 per group).It should be noted that the target dose vastly exceeds targetconcentration in rats, so it is expected that differences in affinitiesbetween parent dAbs and trypsin-resistant dAbs (see example 11) will notimpact on the fate of the molecules in vivo. Hence differences in PKprofiles between dAbs are expected to reflect on an antigen-independentelimination process.

Blood samples were taken after 0.03, 1, 4, 8, 24, 48, 72, 96, 120 and168 hours post administration. After clot formation, serum was withdrawnand then tested in hIL-1R1, TNFR1 or VEGF antigen capture assays:

hIL-1R1Antigen Capture Assays:

Coat with 4 ug/mL anti-hIL-1R1

Block

Add 500 ng/mL shIL-1R1

Add samples

Detect with anti-human Fc HRP@1:10,000

TNFR1Antigen Capture Assays:

Coat with 0.1 ug/mL sTNFR1

Block

Add samples

Detect with anti-human Fc HRP@1:10,000

VEGF Antigen Capture Assays:

Coat with 0.25 ug/mL VEGF

Block

Add samples

Detect with anti-human Fc HRP@1:10,000

Raw data from the assays were converted into concentrations of drug ineach serum sample. The mean μg/mL values at each time point were thenanalysed in WinNonLin using non-compartmental analysis (NCA). The PKprofiles of each dAb-Fc pair are shown in Table 16 which summarises thedetermined PK parameters.

TABLE 16 Half AUC/D (0-inf) Life (hr * μg/mL)/ % AUC ID dAb (hr) (mg/kg)Extrapolated DMS1541 4-130-54 93.2 691.5 22.7 DMS1542 4-130-202 176.8710.1 49 DMS1543 1h-131-511 140.8 1807.5 40 DMS1544 1h-131-206 158.62173.0 43.6 DMS1546 15-10 43.2 324.6 3.8 DMS1531 15-10-11 56.6 770.5n.d. DMS1545 15-26-501 12.9 89 5.1 DMS1529 15-26-593 86.2 804.7 21.0

The results clearly indicate that—whilst the PK profiles of the dAb-Fcpairs 4-130-54 to 1 h-131-206 are almost superimposable—the profilesvary widely with the other pairs. The effects are mostly visible whenAUC/D is considered: the AUC/D of 15-10 is only 42% of that of 15-10-11.The AUC/D of 15-26-501 is only 11% of that of 15-26-593. These importantdifferences also impact (to a lesser extent) half-lives: 43.2 h versus56.6 h for 15-10 and 15-10-11, respectively. A greater difference isseen with the DOM15-26 lineage: 12.9 h versus 86.2h for 15-26-501 and15-26-593, respectively. Indeed for a good PK analysis usingnon-compartmental analysis, there should be at least 4 data points usedto fit the linear regression slope and the period of time over which thehalf life is estimated should be at least 3 times that of the calculatedhalf life.

In light of the biophysical properties described in the examples herein,it appears that the ability of any given dAb to resist degradation bytrypsin is correlated with the ability of the dAb-Fc fusion to circulatefor longer period in the rat serum. Indeed as shown in the examples,such as Example 10, DOM15-10 and DOM15-26-501 are the most degradabledAbs: incubation of 25 ug dAb in the presence of 1 ug of trypsin at 30°C. for ˜3 h resulted in complete degradation. All other dAbs in thisstudy (whether they had been selected with trypsin (ie. DOM15-10-11,DOM15-26-593, DOM4-130-202 and DOM1h-131-206) or whether they alreadyhad some trypsin resistance as parent molecules (DOM4-130-54 andDOM1h-131-511)) have comparable PK profile in rats when re-formattedinto dAb-Fc molecules. Thus, the present PK study suggests thatsusceptibility to proteolysis has its biggest impact on the in vivostability of dAbs when those dAbs have very low resistance toproteolysis. It also shows that—beyond a certain level—furtherincrements in resistance to degradation by trypsin (e.g. DOM4-130-206 vDOM4-130-54) do not significantly add up to the ability of the dAb-Fcmolecule to further slow down elimination in vivo.

In three cases, selection in the presence of trypsin resulted in newmolecules with increased thermal stability (defined by the meltingtemperature): DOM4-130-202, DOM1h-131-206 and DOM15-26-593. The PK studyindicates that—in the present dataset—melting temperature is not anadequate parameter to rationalize the observed PK profiles: indeedDOM15-10 has a higher Tm than DOM15-10-11 and yet is more rapidlycleared than DOM15-10-11 from the circulation. Elsewhere, the two dAbsof the DOM4-130 lineage have markedly different Tm (by 10° C.) and yetshow almost identical stability in vivo when formatted into dAb-Fcmolecules. It should be noted that melting temperature is not per seexcluded as key parameter to predict in vivo stability. It just happensthat with the present dataset, large Tm differences (from 54° C. andabove) have not a significant impact on the fate of dAbs in vivo. Thisdoesn't exclude the possibility that at melting temperature lower than54° C., the in vivo stability of dAbs may correlate with thermalstability, or perhaps even with thermal stability and resistance toproteases altogether.

Example 13 Trypsin Selections on DOM10-53-474

Trypsin Stability of Purified DOM10-53-474:

DOM10-53-474 is a domain antibody which binds to IL-13 with a highpotency. To assess the stability of this dAb in the presence of trypsin,purified dAb was digested with trypsin for increased time points and runon a gel to examine any possible protein degradation. 25 μl of purifiedDOM10-53-474 at 1 mg/ml was incubated with 1 μl of sequencing gradetrypsin at 1 mg/ml at 30° C., resulting in molecular ratio of 25:1dAb:trypsin. dAb was incubated with trypsin for 1 h, 4 h and 24 h andthe protease activity was neutralised by addition of 4 μl of Rochecomplete protease inhibitors followed by incubation on ice. Time 0sample was made by adding protease inhibitors to dAb without addingtrypsin. 2 μl of sample was subsequently analysed by electrophoresisusing Labchip according to manufacturers instructions.

FIG. 22 shows a gel run with DOM10-53-474 incubated with typsin forincreased time points. For comparison one of the trypsin stable dAbs,DOM15-26-593 was also treated with trypsin as explained above and wasrun alongside. As shown in the figure, DOM15-26-593 looks stable evenafter 24 h incubation with trypsin. However, DOM10-53-474 is degraded toa certain extent after 24 h, but looking stable at 1 h and 4 h timepoints. These data suggests that DOM10-53-474 is resistant todegradation by trypsin to a certain extent, but is not as stable as oneof the most trypsin stable dAbs DOM15-26-593.

Trypsin Stability of Phage-Displayed DOM10-53-474:

To assess the trypsin stability of phage displayed DOM10-53-474, thegene encoding DOM10-53-474 was cloned into Sal/Not sites of pDOM33 (FIG.50) and phage produced according to standard techniques. Phage waspurified by PEG precipitation, re-suspended in PBS and titered.

Phage displayed dAbs were incubated with trypsin for different timepoints to evaluate trypsin resistance. Following incubation withtrypsin, stability was measured by titre analysis following infection ofexponentially growing E. coli TG1 cells.

100 μl of phage was incubated in 100 μg/mltrypsin for 1 h, 2 h, 4 h andovernight at 37 C., in a shaking incubator. Trypsin activity was blockedwith Roche complete protease inhibitor (×2) and then phage was dilutedin 2% marvel in PBS, incubated with 10 nM biotinylated IL-13 for onehour at room temperature. Streptavidin-coated beads (Dynabeads M-280(Invitrogen) that were pre-blocked for one hour at room temperature with2% marvel in PBS was added, and the mixture was then incubated for 5minutes at room temperature. All of the incubation steps with Dynabeadswere carried out on a rotating wheel. Unbound phage was washed away bywashing the beads eight times with 1 ml of 0.1% Tween-20 in PBS. Boundphage was eluted with 0.5 ml of 0.1M Glycine pH 2.2 and neutralized with100 μl of 1M Tris-HCL pH 8.0. Eluted phage was used to infectexponentially growing TG1 (1 h at 37° C.) and plated on tetracyclineplates. Plates were incubated at 37° C. overnight and colony counts weremade. Phage output titres following digestion with trypsin is summarisedin Table 17. Phage titres decreased when incubated with trypsin forincreased time points. After 24 h incubation all phage was digested.

TABLE 17 Output titres of trypsin selections performed on phagedisplayed DOM-10-53-474 parent: Length of Trypsin trypsin incubationconcentration Titre No trypsin control — 3 × 10⁷ 1 h 100 μg/ml 1 × 10⁷ 2h 100 μg/ml 7 × 10⁶ 4 h 100 μg/ml 5 × 10⁶ overnight 100 μg/ml 0Selection of dAbs More Resistant to Trypsin:

In order to select for dAbs which are more resistant to degradation bytrypsin, random mutations were introduced to gene encoding DOM10-53-474by PCR using Stratergene Mutazyme 11 kit, biotinylated primers and 5-50pg of template for 50 μl reaction. After digestion with Sal1 and Not1,inserts were purified from undigested products with streptavidin coatedbeads and ligated into pDOM33 at the corresponding sites. E. Coli TB1cells were transformed with purified ligation mix resulting in an errorprone library of DOM10-53-474. The size of the library was 1.9×10⁹ andthe rate of amino acid mutation was 1.3.

Three rounds of selections were performed with this library to selectfor dAbs with improved protease resistance. First round of selection wasperformed only with antigen without trypsin treatment to clean up thelibrary to remove any clones that no longer bound antigen with highaffinity. Selection was carried out at 10 nM IL-13. The outputs fromround one were 2×10⁹ compared to input phage of 6×10¹⁰ indicating thatmajority of library bound antigen with high affinity.

The second and third rounds of selections were performed with 1 nMbiotinylated IL-13. Prior to panning on IL-13, phage was incubated with100 μg/ml of trypsin at 37° C. in a shaker (250 rpm). For second roundselection, trypsin incubation was carried out for 1 h either at roomtemperature or at 37° C. The outputs from round 2 selection is shown inTable 18:

TABLE 18 Output phage titres following second round selection. Trypsintreatment Titre No treatment 1 × 10⁸ 1 h room temperature 5 × 10⁷ 1 h37° C. 2 × 10⁷

Phage outputs from round 2 selection with 1 h trypsin treatment at 37°C. was used as the input for 3^(rd) round selection. For 3^(rd) roundselection, phage was treated with 100 μg/ml trypsin but for longer timepoints: 2 h at 37° C., 4 h at 37° C., overnight at room temperature orovernight at 37° C. The output titres for 3^(rd) round selection aresummarised in Table 19:

TABLE 19 Output phage titres following third round selection Trypsintreatment Titre No trypsin 1.3 × 10⁸ 2 h at 37° C. 1.9 × 10⁷ 4 h at 37°C.   2 × 10⁶ Overnight at room temperature   4 × 10⁷ Overnight at 37° C.2.1 × 10⁶

Several clones from each selection outputs from round 1, 2 and 3 weresequenced to assess the sequence diversity. Following first round ofselection without trypsin treatment, 50% of the selection outputs hadparent DOM10-53-474 sequence. After 2^(nd) round of selection,percentage of parent increased to 75%. After 3^(rd) round of selection,percentage of parent increased to 80%.

This data indicate that DOM10-53-474 is already resistant to degradationby trypsin and not many new clones can be selected from these trypsinselections. FIG. 22 showed that when purified protein was digested withtrypsin, DOM10-53-474 was not completely digested even after overnighttrypsin treatment. However to see whether there are any new clones thatare more trypsin resistant than DOM10-53-474 in selection outputs,selection 3 output where phage was treated overnight with trypsin at 37°C. was sub-cloned into pDOM5. Hundred clones were then sequenced to lookfor any trypsin resistant clones. Out of hundred clones analysed, only26 clones had new sequences, however none of these clones had mutationsat trypsin cleavage sites (Lysine or Arginine) suggesting that theseclones are not more resistant to trypsin than DOM10-53-474.

Example 14 Storage and Biophysical Improvements Introduced into the LeadDOM0101 (Anti-TNFR1) dAbs by Phage Selections in the Presence of Trypsin

To improve the protease resistance of the lead molecule DOM1h-131-511,phage selections in the presence of trypsin were carried out asdescribed earlier. The method produced a range of clones with improvedtrypsin stability compared to the parental DOM1h-131-511 molecule. Twoclones, DOM1h-131-202 and DOM1h-131-206 were selected for furthercharacterisation as they showed the most significant improvement to theaction of trypsin. Further work as outlined below shows that with theimproved resistance to the action of trypsin there are other beneficialeffects, primarily on the stability of the molecules to shear andthermal stress. These two parameters are central to increasing thestorage and shelf life stability of biopharmaceutical products.

Production of Lead DOM0101 dAbs in Pichia pastoris:

The genes encoding the primary amino acid sequence of the three leadmolecules was used to produce secreted protein in Pichia pastoris. Thethree synthetic genes (DOM1h-131-511, DOM1h-131-202 and DOM1h-131-206)were cloned into the expression vector pPIC-Za and then transformed intotwo Pichia strain, X33 and KM71H. The transformed cells were plated outonto increasing concentrations of Zeocin (100, 300, 600 and 900 μg/ml)to select for clones with multiple integrants. Several clones were thenscreened in 2 L flasks to identify high expressing cell lines. The bestexpressing clones were then used to produce material at 5 L scale infermenters.

Protein Purification and Material Characterization:

In order to produce high quality material for characterisation andfurther stability studies, a downstream purification process was devisedusing a mixed modal charge induction resin (Capto MMC) as the primarycapture step followed by anion exchange (Q Sepharose). Withoutsignificant optimisation, this allowed the recovery of ˜70% of theexpressed dAb at a purity of ˜95%. The material was characterised foridentity using electrospray mass spectrometry, amino terminal sequencingand isoelectric focusing and for purity using SDS-PAGE and SEC (sizeexclusion chromatography).

Protein Identity:

The amino terminal sequence analysis of the first five residues of eachprotein, was as expected. Mass spectrometry was performed on samples ofthe proteins which had been buffer exchanged into 50:50 H₂O:acetonitrilecontaining 0.1% glacial acetic acid using C4 Zip-tips (Millipore). Themeasured mass for each of the three proteins was within 0.5Da of thetheoretical mass based on the primary amino acid sequence (calculatedusing average masses) when allowing for a mass difference of −2 from theformation of the internal disulphide bond. IEF was used to identify theproteins based on their pI which was different for each protein.

Protein Purity:

The three proteins were loaded onto non-reducing SDS-PAGE gels in 1 μgand 10 μg amounts in duplicate. A single band was observed in allinstance.

Size exclusion chromatography was also performed to demonstrate levelsof purity. For size exclusion chromatography (SEC) 100 μg of eachprotein were loaded onto a TOSOH G2000 SWXL column flowing at 0.5ml/min. Mobile phase was PBS/10% ethanol. The percentage of monomer wasmeasured based on the area under the curve (see FIG. 23).

Comparison of Stability of DOM1h-131-511, -202 and -206

Assessment of Protease Stability:

The protease stability of DOM1h-131-511, DOM1h-131-202 and DOM1h-131-206was assessed by BIAcore™ analysis of the residual binding activity afterpre-incubation for defined timepoints in excess of proteases.Approximately 1400RU of biotinylated TNFR1 was coated to a streptavidin(SA) chip. 250 nM of DOM1h-131-511, DOM1h-131-202 and DOM1h-131-206 wasincubated with PBS only or with 100 ug/ml of trypsin, elastase orleucozyme for 1, 3, and 24 hour at 30° C. The reaction was stopped bythe addition of a cocktail of protease inhibitors. The dAb/proteasemixtures were then passed over the TNFR1 coated chip using referencecell subtraction. The chip surface was regenerated with 10 ul 0.1Mglycine pH 2.2 between each injection cycle. The fraction ofDOM1h-131-511, DOM1h-131-202 and DOM1h-131-206 bound to human TNFR1 (at10 secs) pre-incubated with proteases was determined relative to dAbbinding without proteases. BIAcore™ runs were carried out at 25° C. Thedata below was produced from three independent experiments. The bargraph indicates mean values and the error bars indicate standarddeviation of the results (FIG. 24).

It was found that DOM1h-131-202 and DOM1h-131-206 were shown to havegreater resistance to proteolytic degradation by trypsin, elastase orleucozyme in comparison to DOM1h-131-511. The difference betweenDOM1h-131-202 and DOM1h-131-206 in comparison to DOM1h-131-511 is mostpronounced after 1 hr with trypsin and after 3 hrs with elastase orleucozyme. There is a trend that DOM1h-131-206 is slightly more stablecompared to DOM1h-131-202 in most of the conditions tested.

Thermal Stability of the dAbs as Determined Using DSC:

In order to determine at which pH the lead molecules had the greateststability, differential scanning calorimeter (DSC) was used to measurethe melting temperatures (T_(m)) of each dAb in Britton-Robinson buffer.As Britton-Robinson is made up of three component buffer systems (40 mMof each of acetic, phosphoric and boric acid), it is possible to producea pH range from 3-10 in the same solution. The theoretical pI wasdetermined from the proteins primary amino acid sequence. From the DSC,the pH at which the dAbs had their greatest intrinsic thermal stabilitywas found to be pH 7 for DOM1h-131-202, pH 7-7.5 for DOM1h-131-206 andpH 7.5 for DOM1h-131-511. For all subsequent stress and stability workthe following pHs were used for each dAb; for DOM1h-131-202 andGSK1995057A DOM1h-131-206 pH 7.0 and for DOM1h-131-511 pH 7.5 inBritton-Robinson buffer. The results are summarised in Table 20.

TABLE 20 Summary of the pH and T_(m)s of DOM1h-131-202, DOM1h-131-206and DOM1h-131-511 as determined by DSC in Britton-Robinson buffer at 1mg/ml. The temperature was ramped at 180° C./hour. pH that givesgreatest Tm (° C.) of the dAb at dAb intrinsic thermal stability thegiven pH DOM1h-131-202 7.0 68.6 DOM1h-131-206 7.0-7.5 65.8 DOM1h-131-5117.5 58.0

Two Week Thermal Stability Testing

The ability of a protein to endure prolonged periods of time at elevatedtemperatures is usually a good indication of its stability. Under theseconditions, protein may undergo several physical processes such asaggregation or chemical modification. The dAbs (at 1 mg/ml) wereincubated at 37 and 50° C. for 14 days in Britton-Robinson buffer. SECwas used to determine how much monomer was left in solution over the 14day period (FIG. 25).

From FIG. 25 it can be seen that both DOM1h-131-202 and DOM1h-131-206are significantly more stable than DOM1h-131-511 to thermal stress.Exposing proteins to elevated temperatures, such as 37 and 50° C., areroutinely used to give an indication on a drug's long term shelf-life.These higher temperatures are used to accelerate the normal processassociated with long term storage at room temperature such asdeamidation, oxidation or aggregation. The level of aggregationformation in solution can also be monitored using SEC (FIG. 26A to I).After 14 days at 37° C., the loss of DOM1h-131-511 from solution can beattributed to both precipitation and the formation of higher orderedaggregates as determined by SEC (FIG. 26B). A significantly lower lossin protein is also seen with both DOM1h-131-202 and DOM1h-131-206 at 37°C. after 14 days with very little or no substantial increase inaggregate formation, especially in the case of DOM1h-131-206 (FIG. 26H).At 50° C., the difference between the molecules is even more pronounced,with DOM1h-131-206 showing better stability at the higher temperaturethan DOM1h-131-202 after 14 days, showing significantly reducedformation of higher molecular weight aggregates (FIG. 26). Relative tothe t=0, DOM1h-131-206 shows only a small increased in aggregateformation after 14 days (FIG. 261), whereas DOM1h-131-511 has all butprecipitated out of solution (FIG. 26C).

This shows that the changes introduced into the dAb by the trypsinselections, e.g. the improved thermal stability, has significantlyimproved the protein storage stability at 37 and 50° C. BothDOM1h-131-202 and more significantly DOM1h-131-206, clearly haveimproved solution stability and lower tendency to form aggregates atelevated temperatures which can directly be translated to improved longterm storage stability at more relevant temperatures such +4° C. androom temperature.

Samples from 24 hr, 48 hr, 7 days and 14 days time points from thethermal stress experiment were then analysed by IEF to see if theproteins had undergone any biophysical changes which would affect theoverall charge of the protein (FIG. 27).

Again both DOM1h-131-202 and DOM1h-131-206 show no significant changesat 37° C. compared to DOM1h-131-511. With DOM1h-131-511a faint secondband appears at 37° C. after 24 hrs. It is believed this extra bandingis due to dimerisation of the protein, thus masking charge and producingtwo populations of molecules. At 50° C. the difference between themolecules is more pronounced, DOM1h-131-206 clearly shows no significantchanges at the elevated temperature whereas DOM1h-131-202 is showingsome sign of modification after 24 hr. The majority of DOM1h-131-511 islost by precipitation after 48 hr in Britton-Robinson.

The T=0, 7 and 14 day time points at 50° C. were analysed by the TNFR-1RBA to determine the functionality of the protein after exposure to hightemperatures (FIG. 28). The assay is currently not as sensitive as SECor IEF at detecting subtle changes to the molecule due to stress, but itcan be used show that the dAb can still bind to the antigen.

The shift in the curve to the left for DOM1h-131-511 reflects the factthat the majority of the dAb has been lost due to precipitation. Thematerial that is left in solution is still able to bind antigen. Asshown in FIG. 25, the majority of both DOM1h-131-202 and DOM1h-131-206are able to be maintained in solution even after 14 days. The RBA showsthat all the soluble protein is still functional and able to bind toTNFR1.

Storage Stability Testing at High Protein Concentrations:

Experiments were carried out to investigate the storage stability at +4°C. at very high protein concentrations to see how each moleculeperformed under these conditions. All the lead dAbs were concentrated incentrifugal Vivaspin concentrators (5K cut-off) in Britton-Robinsonbuffer at their most stable pH, to ˜100 mg/ml. The samples at ˜100 mg/mlwere then left at +4° C. for 7 days and then analysed by SEC to see ifany other physical changes had occurred to the samples during storage athigh concentrations (FIG. 29). The samples were diluted to ˜1 mg/mlbefore being run on the SEC column in 1×PBS10% ethanol (v/v).

From the SEC traces it can be seen that neither DOM1h-131-202 norDOM1h-131-206 show any significant increase in the formation ofaggregates after 7 days, where as there is ˜2% reduction in the monomerconcentration for DOM1h-131-511.

Nebuliser Delivery of the Lead dAbs:

For early stage toxicology and clinical work, the dAbs will beformulated as a liquid and delivered via a nebulising device. Dependingon the device (eg, ultrasonic, jet or vibrating mesh), the dAb willexperience a degree of shear and thermal stress as it was nebulised toform a aerosol of defined particle size. As both DOM1h-131-202 and -206have higher T_(m)'s and showed considerably improved stability tothermal stress compared to DOM1h-131-511, all the dAbs were tested intwo nebuliser devices to see how they responded to shear/thermal stressinduced during nebulisation. Both the protein from the nebulised aerosoland the remaining dAb in the device (i.e. in the cup) were then analysedby SEC to determine the amount of aggregation generated during theprocess.

All the molecules were tested in Britton-Robinson buffer at their moststable pH. The dAbs were tested in both the E-flow Rapid (vibratingmesh) and Pari LC+ (jet nebuliser) with run time of 3.5 minutes at aprotein concentration of 5 mg/ml and the particle size distributiondetermined using a Malvern Spraytek. The results are shown in FIG. 30.For good delivery and distribution into the deep lung, the idealparticle size is <5 μm. All the dAbs give comparable levels of particlesizes that were less than 5 μm in Britton-Robinson buffer. Theconcentration of the dAb in the cup of the device was determined by A₂₈₀measurements before and after nebulisation (data not shown). It wasfound that the protein concentration did not change significantlyindicating that neither the protein nor vehicle are preferentiallynebulised during delivery.

Samples of the dAbs nebulised in Britton-Robinson buffer were run on SECto determine if during delivery the protein had undergone any physicalchanges. FIG. 31 shows the relative percentage change in either the cupor the aerosol as determined by SEC. It can be seen that bothDOM1h-131-202 and DOM1h-131-206 undergo relative small changes in theconcentration of monomer relative to DOM1h-131-511. This demonstratesthat both DOM1h-131-202 and DOM1h-131-206 with their improved T_(m)'shave less propensity to aggregate during nebulisation.

FIG. 32 shows the actual SEC traces for DOM1h-131-206 and DOM1h-131-511in Britton-Robinson buffer post nebulisation and demonstrates that therelative loss in monomer (FIG. 31) is due to dimer formation. This againprovides further supporting evidence to the theory that the greaterthermal stability shown by DOM1h-131-202 and DOM1h-131-206 can preventsignificant aggregation even in an unoptimised formulation buffer.

For toxicology and safety assessment work, it is necessary to deliverythe dAb at significantly higher levels into the animal than thetherapeutic doses given to patients. This can only be achieved by usingsignificantly higher protein concentrations and/or delivering the dAbover a prolonged period of time. As it had already been shown thatDOM1h-131-511 forms aggregates on nebulisation at 5 mg/ml over 3.5 mins,DOM1h-131-206 was tested at 40 mg/ml in PBS and nebulised using the PariLC+ for up to 1 hour. Samples from the cup and aerosol were taken at thetime points to throughout the run to see if the prolong nebulisationcaused the dAbs to aggregate due to shear or thermal stress asdetermined by SEC and the protein concentration (A280 nm measurements).Table 21 shows the protein concentration of the dAb both in the cup andaerosol as determined by A280.

TABLE 21 Measured protein concentration of DOM1h-131-206 as determinedby A280 absorbance readings for both the cup and aerosol duringnebulisation of the dAb at ~40 mg/ml using the Pari LC+. Allowing fordilution errors and instrumental error the sample concentration does notchange after nebulising the dAb over 1 hr. Time Cup Sample AerosolSample (Mins) (mg/ml) (mg/ml) 1 43.8 43.4 29 44.5 43.5 59 44.6 44.1

From Table 21 it can be seen that the concentration of the protein didnot significantly change during the run, demonstrating that there was nosignificant loss of the protein due to aggregation. FIG. 33 shows thatover the period of 1 hour of nebulisation, DOM1h-131-206 does not formany higher ordered aggregates such as dimers as determined by SEC. Thisclearly demonstrates that the improved biophysical properties, asintroduced into the molecule by trypsin selections, significantlyincreases the dAbs resistance to shear and thermal stress and that thiscan be directly correlated to improved storage shelf-life and theability to nebulise the protein so that higher ordered aggregates do notform.

Solution State of the Lead dAbs:

Since the major route of degradation for all the three lead dAbs appearsto be self-association leading initially to dimerisation followed byfurther aggregation and ultimately precipitation, the three leadmolecules were investigated by Analytical Ultra-Centrifugation (AUC) todetermine the degree of self-association. The proteins were investigatedby two methods, sedimentation equilibrium and sedimentation velocity.

For the sedimentation equilibrium method the three samples were run atthree different concentrations ranging from 0.5 mg/ml to 5 mg/ml withcentrifugation effects using three different rotor speeds. By thismethod it was determined that DOM1h-131-511 is a stable dimer (26.1-34.4kDa), DOM1h-131-202 is monomer/dimer equilibrium (22.7-27.8 kDa) with arelatively stable dimeric state at the concentrations measured withK_(d)=1.3 μM and DOM1h-131-206 is predominantly monomeric (15.4-17.9kDa) with a K_(d) for the monomer to dimer association of 360 μM.

By the sedimentation velocity method all samples showed some degree ofdissociation upon dilution. From the results obtained, shown in FIG. 34,the sedimentation coefficient observed for DOM1h-131-511 is indicativeof higher order aggregates and the peak shift upon dilution is anindication of dissociation of these aggregates. The protein aggregationand dissociation cancel each other out which can give the impression ofbeing a stable dimer as observed by sedimentation equilibrium. Thesedimentation coefficients observed for DOM1h-131-202 are indicative ofa rapid dynamic equilibrium and therefore the monomer and dimer peakscould not be separated from each other, giving the single peak with ahigher sedimentation coefficient than is appropriate for the mass of thesample. This result agrees with the result obtained by the sedimentationequilibrium method and the dissociation constant was measured as being 1μM. DOM1h-131-206 was determined to be more monomeric than the other twosamples, having a sedimentation coefficient of 1.9 s as compared to 2.5s for the other two samples. This data agrees well with thesedimentation equilibrium data. At the concentrations measured, ˜10-foldbelow the K_(d) of 360 μM, the sample is predominantly monomeric.

Example 15 Potency Enhancement of the DOM15-26-593 dAb

An example of the enhancement of potency in VEGFR2Receptor Binding Assayof the DOM15-26-593 dAb over DOM15-26 parent is shown in FIG. 40. Inthis assay, the ability of a potential inhibitor to prevent binding ofVEGF to VEGFR2 is measured in a plate-based assay. In this assay aVEGFR2-Fc chimera is coated on a 96-well ELISA plate, and to this isadded a predetermined amount of VEGF that has been pre-incubated with adilution series of the test dAb. Following the washing-off of unboundprotein, the amount of VEGF bound to the receptor is detected with ananti-VEGF antibody, the level of which is determined colorimetrically. Adose-response effect is plotted as percentage inhibition of VEGF bindingas a function of test substance concentration. An effective inhibitor istherefore one that demonstrates substantial blocking of ligand bindingat low concentrations.

FC Fusions Potency and Half Life:

The therapeutic potential of VEGF blockade in the treatment of tumourshas been realised for over 30 years. The chronic nature of cancerdictates that biopharmaceuticals require a long serum half life tomediate their effects, and this is not consistent with the rapidclearance of free dAbs from the circulation by renal filtration. Toassess the utility of the VEGF dAbs as anti-angiogenics for thetreatment of cancer, the lead domain antibodies were formatted asfusions with wild type human IgG1 Fc via a hybrid linker so as to form abivalent molecule with a serum half life extended by the use ofFcRn-mediated antibody salvage pathways.

In this Fc fusion format, the potency of the lead trypsin selected dAb,DOM15-26-593 was compared with the initial parent dAb (DOM15-26) & thetrypsin labile dAb (DOM15-26-501) using the assay described previously.The results are shown in the Table 22 below:

TABLE 22 Potency (RBA) & half life characteristics of DOM15-26 leads inthe Fc fusion format dAb Fc Potency (nM) T½b (hrs) DOM15-26 hIgG1 0.506ND DOM15-26-501 hIgG1 0.323 12.9 DOM15-26-593 hIgG1 0.033 84.6

It can be seen from these results that in the dimeric Fc fusion format,affinity & potency are enhanced in relation to the free dAbs due to theeffect of avidity. It is clear that the potency enhancement obtained inDOM15-26-593 by virtue of trypsin selection is maintained and is evenmore pronounced in this Fc format. Furthermore, the improvements inthermal and protease stability translate into profound changes in the invivo pharmacokinetic behaviour of the molecules. The improvement in theelimination half life (see FIG. 41) of DOM15-26-593 compared withDOM15-26-501 is likely to be a direct consequence of the increasedstability of the dAb, rendering it more resistant to the degradativeprocesses that occur within the endosomal compartment. It is also to beexpected, therefore, that dAbs with enhanced protease stability are ableto persist for longer in other biological compartments such as theserum, mucosal surfaces and various tissue compartments whereproteolysis is an active process involved in the turnover of biologicalmolecules.

Pharmacokinetic Clearance Profiles:

Pharmacokinetic clearance profiles of DOM15-26-593 and DOM15-26-501 weremeasured after i.v. administration DOM15-26-593 and DOM15-26-501 to 3rats at concentrations of 5 mg/kg. Levels of DOM15-26-593 andDOM15-26-501 in the serum were then measured using a direct VEGF bindingstandard ELISA assay and an anti-human Fc antibody, therefore onlyintact drug in the serum samples were detected. The full pharmacokineticprofile is shown in the Table 23 below:

TABLE 23 Summary Pharmacokinetic parameters of the DOM15-26 &DOM15-26-593 Fc fusions in rat Half Life Cmax AUC (0-inf) Clearance dAb(hr) (μg/ml) (hr * μg/ml) (ml/hr/kg) DOM15-26-501 12.9 91.4 445.1 11.8DOM15-26-593 84.6 101.8 3810 1.3

It can be seen from these results that DOM15-26-593 has a significantlyimproved pharmacokinetic profile with e.g. an extended half life andreduce clearance rate.

The significantly improved potency and pharmacokinetic properties of theDOM15-26-593 resulted in analysis of the compound for a range of otherbiophysical attributes.

Solution State Properties: Analysis by SEC-MALLs & AUC:

The in-solution state of DOM15-26-593 was assessed by both sizeexclusion chromatography-multi-angle laser light scattering (SEC-MALLS)and analytical ultracentrifugation (AUC). SEC-MALLS was run on aSuperdex 200 GF column (agarose matrix) at a flow rate of 0.3 ml·min⁻¹in Dulbecco's PBS (Sigma) with refractive index (RI detection on a WyattOptilab rEX) and MALLS detection on a Wyatt TREOS MALLS detector. Datawere analysed using ASTRA software Two separate batches of DOM15-26-593were analysed and both were shown to behave as monomers in solution atconcentrations of up to 2.5 mg/ml with a calculated molecular mass of78-81 KDa, consistent with the calculated intact molecular mass ofapprox 76 kDa.

For the AUC analysis, DOM15-26-593 was diluted to concentrations of 0.2,0.5 and 1.0 mg/ml in PBS & sedimentation velocity runs carried out at40000 rpm in a Beckman XL-A analytical ultracentrifuge. Data wasacquired at 5 minute intervals at a set temperature of 20° C. Data wasanalysed using SEDFIT software and sedimentation coefficientdistributions were generated using either c(S) or ls-g(s*) routines.

The results of this analysis show that DOM15-26-593 behaves as a monomerin solution at concentrations of up to 2.5 mg/ml with a calculatedmolecular mass of 78-81 KDa, consistent with the calculated intactmolecular mass of approx 76 kDa (FIGS. 42 a & 42 b).

Thermal Melting Properties: Analysis by DSC

Experiments were done with DOM15-26-593 (and Fc fusion) as follows:

Differential scanning calorimetry was used to analyse the thermalstability of the dAbs and Fc fusions. Briefly the proteins were placedin a Microcal calorimeter at a concentration of 2 mg·ml⁻¹ with a bufferreference. The samples were heated from 20° C. to 100° C. at a rate of180° C.·hr⁻¹ in an appropriate buffer, and the thermal denaturation dataanalysed using “Origin” software using fitting models appropriate to thenature of the protein under analysis.

The increased thermal stability of the trypsin selected dAb (65° C.,FIG. 43 middle panel) is maintained in the Fc fusion (64.5° C., FIG. 43upper panel). The Tm curve of the DOM15-26-501 dAb (52° C., FIG. 43lower panel) is shown for comparison.

Stability to Freeze-Thaw, Temperature Stress and Serum Components

Experiments were done with DOM15-26-593 (and Fc fusion) as follows:

The stability properties of the DOM15-26-593 dAb mean that it can besubjected to physical and biological stress with minimal effects on itsability to bind VEGF (see FIGS. 44-47 (a and b)). The binding of theVEGF dAb-Fc fusions to VEGF was in all cases determined by ELISA.Briefly, a 96-well ELISA plate was coated with 250 mg/ml VEGF₁₆₅ incarbonate buffer overnight. The plate was then blocked with 1% BSA inPBS prior to addition of the test substances diluted in the same buffer.The unbound material was washed away after 60 minutes incubation, andthe bound material detected with a 1:10,000 dilution of HRP-conjugatedanti-human IgG followed by “SureBlue” chromogenic substrate and stoppingwith 1M HCl.

For example, the molecule can be repeatedly freeze thawed from liquidnitrogen (−196° C.) to body temperature (37° C.) for 10 cycles withoutloss of binding activity as determined by ELISA (FIG. 44). Thistreatment also resulted in no obvious alterations in the molecule'saggregation state, as assessed by conventional size exclusionchromatography (FIG. 45). Further tests demonstrated that the moleculecan be placed at a range of different temperatures from −80° C. to 55°C. with only a minor drop in antigen binding activity after 168 hours atonly the highest incubation temperature (FIG. 46). Furthermore,incubation with serum from human or cynomolgus monkeys at 37° C. for 14days caused no loss of antigen binding ability (FIGS. 47 a and 47 b), asdetermined by the VEGF binding ELISA

Potency in VEGFR2Receptor Binding Assay & HUVEC Cell Assay:

The receptor binding assay described above was used to assess thepotency of the DOM15-26-593 dAb-Fc fusion (FIG. 48). It was found thatthe DOM15-26-593 dAb has enhanced potency in this assay, whichestablishes the ability of the dAb to block the binding of VEGF toVEGFR2 in vitro. The potency of the DMS1529 was also demonstrated in aHUVEC (Human Umbilical Vein Endothelial Cell) assay, where the abilityof VEGF antagonists to block the VEGF stimulated proliferation of HUVEcells is measured. Briefly, approximately 4e3 HUVE cells are dispensedinto the wells of a 96-well plate to which is added a mixture of VEGFand a dilution series of the test substance, such that the finalconcentration of VEGF is 5 ng/ml, or as otherwise determined by adose-response titration. The cells are incubated at 37° C. for a further4 days, at which point the cell number is determined by the use of acell quantification reagent such as “CellTiter”. This allows thecolorimetric determination of cell proliferation in comparison withstandards over the 4 days of the experiment. Cell numbers are determinedat the end of a fixed incubation period with a pre-determined amount ofVEGF and a varying amount of test article. The more potent theantagonist, the lower the cell proliferation observed (FIG. 49).

The invention claimed is:
 1. An anti-VEGF immunoglobulin single variabledomain comprising an amino acid sequence that is identical to the aminoacid sequence of (SEQ ID NO: 238 (DOM 15-26-593).
 2. An anti-VEGFantagonist comprising an anti-VEGF immunoglobulin single variable domainencoded by a nucleic acid sequence that is identical to the sequence ofSEQ ID NO: 239 (DOM 15-26-593).
 3. An anti-VEGF antagonist comprising ananti-VEGF immunoglobulin single variable domain comprising an amino acidsequence that is identical to the amino acid sequence of (SEQ ID NO: 238(DOM 15-26-593).
 4. The anti-VEGF antagonist of claim 3, wherein theantagonist comprises a monomer of said single variable domain or ahomodimer of said single variable domain.
 5. A composition comprisingthe immunoglobulin single variable domain of claim 1, and apharmaceutically acceptable carrier, excipient or diluent.
 6. Acomposition comprising the antagonist of claim 3 or 4, and apharmaceutically acceptable carrier, excipient or diluent.